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https://openalex.org/W3165621877	https://bmcmusculoskeletdisord.biomedcentral.com/track/pdf/10.1186/s12891-021-04377-4	English	null	Duloxetine reduces pain after Total hip arthroplasty: a prospective, randomized controlled study	BMC musculoskeletal disorders	2,021	cc-by	6,247	Duloxetine reduces pain after Total hip arthroplasty: a prospective, randomized controlled study Li1†, Wei-Nan Zeng1,2†, Zi-Chuan Ding1, Ming-Cheng Yuan1, Yong-Rui Cai1 and Zong-Ke Zhou1* Abstract Background: Previous studies have demonstrated the efficacy of duloxetine in reducing postoperative pain and opioid consumption. However, the effect of duloxetine on total hip arthroplasty (THA) remains unclear. The objective of this study was to assess the efficacy of oral duloxetine in THA. Methods: We enrolled 96 patients in this randomized controlled trial. These patients were randomized (1,1) to either the duloxetine group or the placebo group and received daily doses of 60 mg duloxetine or placebo, respectively, from 2 d pre-operation to 14 d after surgery. The primary outcome was pain severity upon movement measured by a visual analogue scale (VAS). The secondary outcomes included VAS scores for resting pain, morphine consumption, Harris Hip Score, patient satisfaction at discharge, length of postoperative hospital stay, and adverse events. Results: Patients in the duloxetine group had significantly lower pain severity scores upon movement within 3 postoperative weeks (p < 0.05) while none of the differences met the minimum clinically important difference (MCID). Moreover, patients in the duloxetine group performed better in terms of resting pain (in 3 weeks after surgery), morphine requirements, and satisfaction level at discharge (all p < 0.05). There was no difference between groups in the prevalence of adverse events. Conclusions: Although it did not result in a clinically meaning reduction in pain after total hip arthroplasty, perioperative administration of 60 mg of duloxetine daily significantly alleviated pain in the postoperative 3 weeks and morphine requirements during the postoperative 48 h. Therefore, duloxetine still shows promise in optimizing the multimodal pain-management protocols in total hip arthroplasty. Trial registration: Chinese Clinical Trial Registry, ChiCTR2000033606, 06/06/2020. Trial registration: Chinese Clinical Trial Registry, ChiCTR2000033606, 06/06/2020. Keywords: Duloxetine, Total hip arthroplasty, Postoperative pain * Correspondence: zhouzongke@scu.edu.cn * Correspondence: zhouzongke@scu.edu.cn †Hao Li and Wei-Nan Zeng contributed equally to this work. 1Department of Orthopaedics, West China Hospital, Sichuan University, 37# WuhouGuoxue Road, Chengdu, China Full list of author information is available at the end of the article * Correspondence: zhouzongke@scu.edu.cn †Hao Li and Wei-Nan Zeng contributed equally to this work. 1Department of Orthopaedics, West China Hospital, Sichuan University, 37# WuhouGuoxue Road, Chengdu, China Full list of author information is available at the end of the article Li et al. BMC Musculoskeletal Disorders (2021) 22:492 https://doi.org/10.1186/s12891-021-04377-4 Li et al. BMC Musculoskeletal Disorders (2021) 22:492 https://doi.org/10.1186/s12891-021-04377-4 Open Access Methods A prospective study, in the form of a single-center (West China Hospital of Sichuan University), randomized, double-blinded, parallel-arm, placebo-controlled clinical trial, was conducted. In this study, we enrolled 153 pa- tients who were scheduled for THA from June 2020 to September 2020. The study was approved by the Ethics Committee on Biomedical Research, West China Hospital of Sichuan University (approval no. 2020–843) and registered at the Chinese Clinical Trial Registry (ChiCTR2000033606, 06/06/2020). Informed consent and research authorizations were obtained from all par- ticipants. The research report met unified clinical trial reporting standards and conformed with the Declaration of Helsinki [16]. Approximately 7 to 23% of THA patients suffer postoperative pain, which is one of the most signifi- cant unfavorable outcomes related to this procedure [3, 4]. Postoperative pain seriously affects periopera- tive mood, interferes with joint function recovery, prolongs hospitalization, increases medical expenses, and further reduces the quality of life and work [5]. Inadequate perioperative pain management is associ- ated with a distinct possibility of suffering postopera- tive pain and numerous other serious complications such as joint stiffness, deep vein thrombosis, and pul- monary embolism [5–8]. Eligible patients who were over 18 years of age; classi- fied under American Society of Anesthesiologists (ASA) status I, II, or III; and scheduled to undergo primary THA for end-stage hip joint diseases, were screened using the Hamilton Depression Scale (HAMD) and the Hamilton Anxiety Scale (HAMA). Subjects whose HAMD and HAMA scores were both < 7 were included. The exclusion criteria were as follows: a known allergy to any of the studied drugs, previous exposure to SNRIs or selective serotonin reuptake inhibitors; known psychi- atric disorders; alcohol or opioid abuse; acute infections of the hip joint; recent treatment for malignant diseases; major previous ipsilateral hip arthroplasty or open sur- gery; peripheral or central nerve impairment; cognitive dysfunction; history of peptic ulcers or bleeding ten- dency; impaired liver and/or renal function; and poor physical condition indicating the lack of ability to toler- ate surgery. Severe postoperative pain exacerbates opioid usage and subsequent opioid-related deaths [6]. In 2017, death rates related to overdosing on opioids, including metha- done and heroin, rose sharply to 19.3 per 100,000 in the United States [9]. Opioid prescriptions dispensed by orthopedic doctors accounted for a considerable per- centage (7.7%) of prescriptions [10]. Background Total hip arthroplasty (THA) is a common surgical pro- cedure used to successfully, economically, and safely treat end-stage joint diseases that may cause deformities, lower the quality of life, and lead to bodily dysfunction [1]. Despite excellent outcomes, the overall incidence of dissatisfaction associated with THA is relatively high, at approximately 20% [2]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. BMC Musculoskeletal Disorders Li et al. BMC Musculoskeletal Disorders (2021) 22:492 Page 2 of 8 Li et al. BMC Musculoskeletal Disorders (2021) 22:492 whether duloxetine optimizes perioperative analgesia protocols in THA. Results A total of 153 patients with end-stage joint diseases were scheduled to undergo a primary unilateral THA proced- ure during the recruitment period (June 2020 to Septem- ber 2020) at our department. Among these 153 patients, 49 did not satisfy the inclusion criteria and 8 declined to participate in the study. Thus, 96 eligible patients were included in the study. These patients were randomly assigned to either the duloxetine group or the placebo group. Specific information regarding patient flow is plotted (Fig. 1). There were no significant differences in the preoperative demographics and characteristics of the study participants between these groups (Table 1; p > 0.05). The pain severity scores upon movement within the 3 postoperative weeks in patients in the duloxetine group were significantly lower compared to those in the pla- cebo group (Table 2; p < 0.05). However, none of the between-group differences exceeds the MCID, and there was no significant difference between the pain severity scores upon movement in patients of the duloxetine and placebo groups after 3 months of surgery. Similar results were obtained for resting pain, wherein the post-operative movement-evoked pain level in pa- tients of the duloxetine group was significantly lower than those in the placebo group until postoperative week 3 (Table 3; p < 0.05). There were no significant differ- ences between the VAS scores for resting pain of the 2 groups after 3 months of surgery. Postoperative mor- phine consumption and HSS until 24 h, 72 h, and 1w after surgery are shown in Table 4. Patients in the dulox- etine group required significantly less morphine com- pared to those in the placebo group (p < 0.05). There was no significant difference in HSS between the duloxe- tine group and the placebo group at either 21 d or 3 months after surgery. In terms of the satisfaction level at discharge, 45 patients in the duloxetine group expressed satisfaction (defined as extremely, very, or somewhat sat- isfied) with the treatment, compared to 36 patients in The pain severity score upon movement (3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 1w, 3w, and 3 m following surgery) was considered as the primary outcome, because movement- evoked pain during the postoperative period is more se- vere, more frequent, and exerts a greater influence on postoperative functional rehabilitation compared with pain at rest [22]. Methods Various multimodal analgesia regimens have been proposed to alleviate post- operative pain and reduce side effects during the use of postoperative opiates. Further optimization of periopera- tive pain management has always been a major topic of related research efforts [6, 7]. Duloxetine (Cymbalta) is a selective serotonin and norepinephrine reuptake inhibitor (SNRI), approved by the Food and Drug Administration (FDA) for the treat- ment of depression, generalized anxiety disorder, and chronic musculoskeletal pain (osteoarthritis, fibromuscu- lar pain, and chronic back pain, among others) [11, 12]. Duloxetine, which promotes the downregulation of in- hibitory pain pathways in the central nervous system, effectively ameliorates pain associated with hyperexcit- ability corresponding to peripheral sensitization, as well as central sensitization (CS), caused by chronic joint pain [12, 13]. Sample size estimation was determined based on pre- vious studies [12, 17]. A sample size of 48 patients in each group was required to test this 2-tailed hypothesis at a power of 0.80, an alpha level of 0.05, and a dropout rate of 20% for detecting a 2-point difference in the pain severity score between groups following surgery. The pain severity score was evaluated using a visual analog scale (VAS) consisting of a horizontal line divided into 10 equal parts. The ends of the horizontal line were marked “0” and “10” and were used to represent no pain to severe pain, respectively; the middle area represented different degrees of pain. The 2-point difference was de- termined to be the minimum clinically important differ- ence (MCID) because the average acceptable VAS pain score difference following surgery was approximately 2 points according previous studies [12, 18, 19]. However, as far as we know, all previous studies inves- tigating the effects of duloxetine on pain following arthroplasty are focused on total knee arthroplasty (TKA) [12, 14, 15]. To the best of our knowledge, none of the studies have investigated whether duloxetine alle- viates postoperative pain following THA. Based on the hypothesis that duloxetine significantly relieves postop- erative pain following THA and leads to further positive postoperative outcomes, we conducted a randomized, double-blinded, placebo-controlled study to determine Participants were randomized (1,1) to either the dulox- etine group or the placebo group. Randomization was Li et al. BMC Musculoskeletal Disorders (2021) 22:492 Li et al. BMC Musculoskeletal Disorders (2021) 22:492 Page 3 of 8 Li et al. Methods BMC Musculoskeletal Disorders Page 3 of 8 Page 3 of 8 satisfaction at discharge, length of postoperative hospital stay and adverse events. Participants were requested to complete a 7-point satisfaction questionnaire before dis- charge [23]. Satisfaction levels ranged from being ex- tremely satisfied to extremely dissatisfied. Adverse events were recorded until the last day on which duloxe- tine or the placebo was administered. concealed from researchers as well as from the patients, by way of sealed envelopes delivered following hospitalization. A sealed, opaque envelope, containing the randomized grouping plan was prepared in advance. After an eligible patient was assigned a sickbed and ex- cluded from surgical contraindications, an independent researcher opened the randomized envelope in the order in which the patients were enrolled to determine the grouping of that patient. Neither the participants nor the primary investigator was aware of the grouping status until the data analysis stage at the end of the study. The hospital pharmacy prepared two types of indistinguish- able capsules containing either 60 mg of duloxetine or a placebo (starch) for daily oral administration starting from 2 d pre-surgeryto 14 d post-surgery. Surgery was performed by the same senior doctor, an experienced surgeon who had performed over 300 THA annually. All participants accepted a multimodal and standardized an- algesic strategy. From preoperative day 2 to the day be- fore surgery, every patient was given celecoxib 200 mg twice a day (one dose after breakfast and one dose after dinner) for preemptive analgesia. During the operation, all patients received general anesthesia, which was com- posed of an induction of sufentanil 0.5 μg/kg, midazolam 0.04 mg/kg, propofol 1–2 mg/kg and cistracurium 2 μg/ kg intravenously, and a following continuous intraven- ous infusion of 0.1–0.3 μg/(kg•min) of remifentanil, 2–5 mg/(kg•h) of propofol and inhalation of sevoflurane to maintain anesthesia. Besides, all patients were treated with an 80 mL periarticular injection of 0.25% ropiva- caine for local infiltration analgesia. Since postoperative day 1, every patient restarted oral administration of cele- coxib (200 mg twice a day) until 2 weeks after the sur- gery. When acute pain was unbearable or VAS was > 6, morphine (5 mg intravenously) was used as a rescue an- algesic [20, 21]. In the perioperative period, there was no other oral analgesics except celecoxib, and intravenous morphine was the only rescue analgesic before dis- charge. Methods Patients’ discharge criteria for postoperative pain included: pain must be tolerable without affecting daily life and rehabilitation, and the severity of acute pain no longer required intravenous morphine to relieve. All par- ticipants were followed up for 3 months. All data management and statistical analyses were con- ducted using IBM, SPSS version 22.0 software. Whereas the independent t-test was used to analyze differences between continuous variables, such as body mass index (BMI) and age, the chi-square test or Fisher’s exact test was used to analyze categorical variables. The signifi- cance level was set at p < 0.05. Results Secondary outcomes included pain se- verity scores at rest (3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 1w, 3w, and 3 m following surgery), morphine consumption (24 h, 72 h, and 1w following surgery), Harris Hip Score (HHS; 3w and 3 m following surgery), patient Li et al. BMC Musculoskeletal Disorders Li et al. BMC Musculoskeletal Disorders (2021) 22 Page 4 of 8 (2021) 22:492 al. BMC Musculoskeletal Disorders (2021) 22:492 Fig. 1 Schematic of the study design Fig. 1 Schematic of the study design randomized, controlled study to validate the effect of duloxetine on THA. the placebo group (94% versus 75%, p = 0.011). No sig- nificant difference was observed between the lengths of postoperative hospital stay between the two groups. There was no significant difference in the adverse event between groups (Table 5; p > 0.05). Pain, which follows arthroplasty, is a serious complica- tion that always confounds orthopedists. Most recent lit- erature indicates that long-lasting, intense, harmful pain stimuli induced by chronic joint diseases may trigger peripheral nociceptors and upregulate the excitability as well as synaptic efficacy of neurons in the central noci- ceptive pathways, leading to central sensitization. Cen- tral sensitization manifests as pain hypersensitivity and further triggers severe postoperative pain following arthroplasty [12, 13, 24–26]. Because serotonin signaling is involved in pain processing [27], the efficacy of SNRIs, including duloxetine, in resolving postoperative pain has been investigated [15]. Discussion In this trial, perioperative daily oral administration of 60 mg duloxetine from 2 d pre-operation to 14 d after surgery resulted in the low- ering of postoperative movement-evoked pain and rest- ing pain as well as in morphine requirements, while the pain differences between two groups are below the MCID. Moreover, patients expressed an increased satis- faction level at discharge from the hospital. Duloxetine did not appear to increase the incidence of adverse events. In this study, we found that perioperative administra- tion of duloxetine effectively relieved movement-evoked pain and resting pain 3 weeks post-surgery. These results are partially substantiated by a previously conducted randomized controlled trial, in which 80 patients sched- uled for TKA, who were treated with duloxetine, achieved a better analgesic effect during the postopera- tive period from 2 to 12 weeks [12]. Except for the in- trinsic difference between THA and TKA, the reason for the difference between the postoperative periods re- quired to relieve pain may be partly attributed to the dif- ferences in the duration of the oral administration of The use of oral duloxetine in arthroplasty has been studied previously. Some of these studies have demon- strated its efficacy in reducing postoperative pain and opioid consumption [12, 14, 15]. As far as we know, all studies that have been conducted to determine the effi- cacy of duloxetine in alleviating residual pain following total joint replacement have focused on TKA and not on THA. Thus, the effect of orally administered duloxetine on THA remains unclear. Therefore, in an attempt to resolve the aforementioned issue, we conducted a Li et al. BMC Musculoskeletal Disorders (2021) 22:492 Li et al. Discussion BMC Musculoskeletal Disorders Page 5 of 8 Table 1 Preoperative demographics and characteristics Demographic data Agea (yr) 52.7 ± 12.0 50.2 + 13.2 0.333 Male sexb (no.[%] of patients) 22 (46%) 24 (50%) 0.683 BMIa (kg/m2) 24.0 ± 2.9 23.9 ± 3.4 0.890 ASA statusb (no.[%] of patients) I 8 (17%) 4 (8%) II 28 (58%) 32 (67%) III 12 (25%) 12 (25%) Surgical site (right/left) 27/21 24/24 Diagnosisb (no.of patients) Osteonecrosis of femoral head 16 (33%) 17 (35%) Primary osteoarthritis 15 (31%) 13 (27%) Developmental dysplasia of the hip 9 (19%) 10 (21%) Others 8 (17%) 8 (17%) Preoperative parametersa hip function Flexion (°) 91.7 ± 11.8 90.2 ± 15.8 0.610 Abduction (°) 21.5 ± 7.3 21.6 ± 8.4 0.948 Harris Hip Score (points) 42.1 ± 9.2 41.2 ± 10.6 0.652 BPI-pain severity score Average 5.4 ± 1.3 5.3 ± 1.1 0.666 Worst 7.6 ± 1.4 7.2 ± 1.7 0.128 Least 2.7 ± 1.2 2.7 ± 1.1 0.861 Current 5.4 ± 1.6 5.1 ± 1.2 0.243 BPI-interference score General activity 6.5 ± 2.0 6.5 ± 2.3 0.925 Walking 7.3 ± 1.9 7.4 ± 2.5 0.853 Work 6.5 ± 2.1 7.3 ± 2.3 0.082 Sleep 5.0 ± 2.1 4.5 ± 2.3 0.264 Relations with others 5.0 ± 2.3 4.7 ± 1.6 0.408 Enjoyment of life 4.8 ± 1.5 4.5 ± 1.6 0.430 Mood 5.2 ± 2.0 5.5 ± 2.4 0.512 HAMD 3.3 ± 1.3 3.0 ± 1.4 0.246 HAMA 3.4 ± 1.4 3.2 ± 1.4 0.425 BPI Brief Pain Inventory aData are given as the mean ± standard deviation bData are given as the number (percentage) of patients daily was effective in treating painful neuropathy or chronic pain, whereas daily doses lower than 60 mg were ineffective [14, 15, 28]. Clinical pharmacokinetic studies reveal that duloxetine achieves maximum plasma con- centration approximately 6 h after dosing and that its biological half-life is approximately 10–12 h [29]. There- fore, the protocol used for the perioperative administer- ing of duloxetine in our study is believed to be duloxetine as well as its dosage. In our study, patients in the duloxetine group were administered duloxetine starting 2 d pre-surgery to 14 d post-surgery, as opposed to a previous study, which followed a protocol of admin- istering 30 mg of duloxetine orally on the night before surgery and 30 mg per day for 6 weeks post-surgery. Discussion According to previous studies, we used an MCID for the VAS score of 2 in our study, while there were also other studies that determined MCID as other values [12, 18, 19]. To re- duce the occurrence of side effects or complications, an analgesia protocol should preferably be multimodal [31]. When using a multimodal analgesia protocol, it is raising the bar very high to identify the performance of individ- ual intervention, therefore, the findings which did not reach the MCID should have a role in a multimodal pain control protocol [31–33]. In a prospective, randomized controlled study comparing local infiltration anesthetic and control, authors found that the local analgesia group had a significantly lower mean VAS score for pain dur- ing exercise than did the control group (4.7 vs 6.6) on [ ] The findings of our study indicated that duloxetine reduced morphine consumption within 1 w post- THA. Two previous randomized controlled trials that enrolled 106 and 50 participants, respectively, also showed similar results [14, 15]. In one of the former studies, a daily oral dose of 60 mg duloxetine was ad- ministered to patients approximately 30 min prior to them being transferred to the operating room. The treatment was continued until 2 weeks after surgery and was found to significantly reduce the total opioid requirements for over a period of 3 months [15]. The other study revealed that two oral doses of 60 mg duloxetine, administered 2 h before surgery and on the first day after surgery, reduced morphine con- sumption in the first 48 h post-TKA [14]. The incon- sistencies observed during the postoperative periods that warranted the reduction of opioid requirements in these two studies may be owing to the differences in assessment times as well as the dosage of duloxe- tine administered, which in turn are associated with the half-life of duloxetine. Discussion The Cochrane database review and other published literature have indicated that 60 mg of duloxetine administered Li et al. BMC Musculoskeletal Disorders (2021) 22:492 Li et al. BMC Musculoskeletal Disorders ( Page 6 of 8 Table 2 Primary outcomes regarding VAS scores upon the movement Time after surgery Duloxetine* (n = 48) Placebo* (n = 48) P value† 3 h 6.0 ± 0.9 6.5 ± 1.2 0.029 6 h 5.9 ± 0.8 6.4 ± 1.0 0.016 12 h 5.4 ± 0.9 5.9 ± 1.0 0.010 24 h 5.0 ± 1.0 5.5 ± 1.2 0.033 48 h 4.4 ± 1.0 4.8 ± 1.2 0.041 72 h 3.9 ± 0.9 4.5 ± 1.1 0.004 1w 2.8 ± 1.0 3.5 ± 1.1 < 0.001 3w 1.9 ± 1.0 2.5 ± 1.1 0.007 3 m 1.6 ± 1.0 1.8 ± 1.0 0.469 The values are given as the mean and standard deviation. †P values are calculated by independent t-test. P values indicating a significant difference among groups are in bold Table 2 Primary outcomes regarding VAS scores upon the movement Table 4 Secondary outcomes regarding morphine consumption and HHS Duloxetine (n = 48) Placebo* (n = 48) P value† Morphine Consumption PO 24 h 11.0 ± 4.9 14.2 ± 5.9 0.006 PO 72 h 16.8 ± 6.1 20.4 ± 9.8 0.032 PO 1w 18.7 ± 7.3 23.3 ± 13.6 0.039 HHS PO 3w 75.4 ± 5,5 73.7 ± 6.1 0.156 PO 3 m 87.2 ± 4.7 87.8 ± 4.4 0.517 PO postoperative. The values are given as the mean and standard deviation. †P values are calculated by independent t-test. P values indicating a significant difference among groups are in bold Table 4 Secondary outcomes regarding morphine consumption and HHS PO postoperative. The values are given as the mean and standard deviation. †P values are calculated by independent t-test. P values indicating a significant difference among groups are in bold The values are given as the mean and standard deviation. †P values are calculated by independent t-test. P values indicating a significant difference among groups are in bold the first day after surgery (p = 0.008), and the difference for the VAS score of 1.9 offered improved pain control [31]. appropriate and adequate. MCID is an important con- cept to put in perspective statistically significant results that may not be clinically relevant [30]. The values are given as the mean and standard deviation. †P values are calculated by independent t-test. P values indicating a significant difference among groups are in bold Discussion Table 3 Secondary outcomes regarding VAS scores for resting pain Time after surgery Duloxetine* (n = 48) Placebo* (n = 48) P value† 3 h 4.1 ± 1.0 4.5 ± 1.1 0.026 6 h 4.0 ± 1.0 4.4 ± 1.0 0.032 12 h 3.3 ± 1.0 3.8 ± 1.1 0.044 24 h 3.0 ± 1.1 3.5 ± 1.1 0.046 48 h 2.3 ± 1.0 2.8 ± 1.0 0.019 72 h 1.8 ± 0.9 2.2 ± 1.0 0.023 1w 1.0 ± 0.8 1.3 ± 0.8 0.040 3w 0.8 ± 0.7 1.1 ± 0.7 0.043 3 m 0.6 ± 0.5 0.7 ± 0.6 0.851 *The values are given as the mean and standard deviation. †P values are calculated by independent t-test. P values indicating a significant difference among groups are in bold Table 3 Secondary outcomes regarding VAS scores for resting pain The decision to use 60 mg of duloxetine as the daily dose in our study was based on the reports of previ- ous studies [14, 15, 28]. Adequate quality-based evi- dence indicates that a daily dose of 60 mg of duloxetine is indeed the appropriate dosage [28]. Lower daily doses of duloxetine are not efficacious in alleviating pain; moreover, higher daily doses do not improve its efficacy and may even result in adverse events [28]. Our results also demonstrated that dulox- etine did not increase the incidence of adverse events at a daily dose of 60 mg. Our study had a few limitations. First, the 3-month follow-up time may obscure the long-term safety of duloxetine. However, the biological half-life of Li et al. Conclusions This work was supported by 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYJC18039) and Regional Innovation & Cooperation program of Science & Technology Department of Sichuan Province (No. 2021YFQ0028). In conclusion, the perioperative daily administration of 60 mg duloxetine to patients undergoing primary unilat- eral total hip arthroplasty alleviated movement-evoked pain 3 weeks post-surgery, although it did not result in a clinically meaning reduction in postoperative pain. Duloxetine also alleviated resting pain within 3 postop- erative weeks, reduced morphine requirements following surgery, and improved satisfaction levels at discharge, without increasing the incidence of adverse events. Con- sidering the positive results of the current study, it may be concluded that duloxetine shows potential as a novel Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors’ contributions HL d d d HL: study design and writing; WNZ: data collections and writing; ZCD: data collections and data analysis; MCY: data collections and data analysis; YRC: writing; ZKZ: study design. All authors read and approved the final manuscript. Acknowledgements Acknowledgements Not applicable. Discussion BMC Musculoskeletal Disorders (2021) 22:492 Page 7 of 8 Table 5 Secondary outcomes regarding satisfaction level, length of PO hospital stays and adverse events Duloxetine (n = 48) Placebo (n = 48) P value Satisfaction level† (no.[%] of patients) Extremely satisfied 19 (40%) 11 (23%) Very satisfied 21 (44%) 17 (35%) Somewhat satisfied 5 (10%) 8 (17%) Neither satisfied nor dissatisfied 2 (4%) 6 (13%) Somewhat dissatisfied 1 (2%) 4 (8%) Very dissatisfied 0 2 (4%) Extremely dissatisfied 0 0 length of PO hospital staysa (h) 63.4 ± 17.6 70.7 ± 23.7 0.089 Adverse Events† (no.[%] of patients) Nausea and vomiting 8 (17%) 7 (15%) 0.779 Dry mouth 4 (8%) 2 (4%) 0.677 Insomnia 5 (10%) 6 (13%) 0.749 Somnolence 6 (13%) 3 (6%) 0.486 Constipation 7 (15%) 9 (19%) 0.584 Dizziness 3 (6%) 4 (8%) 1.000 Fatigue 5 (10%) 7 (15%) 0.537 aData are given as the mean ± standard deviation. †Data are given as the number (percentage) of patients therapeutic agent and could likely be used to optimize multimodal pain management protocols in total hip arthroplasty. duloxetine is approximately 10–12 h, and maximum plasma concentration is attained approximately 6 h after dosing [29]. Patients with impaired liver and renal function were excluded during screening; there- fore, the 3-month follow-up period was adequate for observing and treating adverse events. A second limi- tation was that the trial was performed at a single center, which may reduce generalizability. Neverthe- less, generalizability is also influenced by different fac- tors such as centers and individuals, as well as by inclusion and exclusion criteria. Lastly, the initial sample size estimation was based on our primary out- come, implying that this sample size may not be ap- propriate for determining a significant difference in terms of all relevant outcomes. Abbreviation THA: Total hip arthroplasty; SNRI: Selective serotonin and norepinephrine reuptake inhibitor; FDA: Food and Drug Administration; CS: Central sensitization; TKA: Total knee arthroplasty; ASA: American Society of Anesthesiologists; HAMD: Hamilton Depression Scale; HAMA: Hamilton Anxiety Scale; VAS: Visual analog scale; MCID: Minimum clinically important difference; HHS: Harris Hip Score; BMI: Body mass index References Lunn MP, Hughes RA, Wiffen PJ. Duloxetine for treating painful neuropathy, chronic pain or fibromyalgia. Cochrane Database Syst Rev. 2014;1:Cd007115. 28. Lunn MP, Hughes RA, Wiffen PJ. Duloxetine for treating painful neuropathy, chronic pain or fibromyalgia. Cochrane Database Syst Rev. 2014;1:Cd007115. 29. Knadler MP, Lobo E, Chappell J, Bergstrom R. Duloxetine: clinical pharmacokinetics and drug interactions. Clin Pharmacokinet. 2011;50(5): 281–94. 29. Knadler MP, Lobo E, Chappell J, Bergstrom R. Duloxetine: clinical pharmacokinetics and drug interactions. 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A novel opioid-sparing pain management protocol following total hip arthroplasty: effects on opioid consumption, pain severity, and patient-reported outcomes. J Arthroplast. 2019;34(11):2669–75. 28. Author details 1 20. Ding ZC, Xu B, Liang ZM, Wang HY, Luo ZY, Zhou ZK. Limited influence of comorbidities on length of stay after total hip arthroplasty: experience of enhanced recovery after surgery. Orthop Surg. 2020;12(1):153–61. 1Department of Orthopaedics, West China Hospital, Sichuan University, 37# WuhouGuoxue Road, Chengdu, China. 2Department of Orthopaedics, 1Department of Orthopaedics, West China Hospital, Sichuan University, 37# WuhouGuoxue Road, Chengdu, China. 2Department of Orthopaedics, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing, China. Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing, China. 21. Li D, Wang Q, Zhao X, Luo Y, Kang P. Comparison of intravenous and topical dexamethasone for total knee arthroplasty: a randomized double- blinded controlled study of effects on dexamethasone administration route and enhanced recovery. J Arthroplast. 2020:1237-43. Received: 18 February 2021 Accepted: 18 May 2021 Competing interests Competing interests The authors declare that they have no competing interests. Publisher’s Note 12. Koh IJ, Kim MS, Sohn S, Song KY, Choi NY, In Y. Duloxetine reduces pain and improves quality of recovery following total knee arthroplasty in centrally sensitized patients: a prospective, randomized controlled study. J Bone Joint Surg Am. 2019;101(1):64–73. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 13. Blikman T, Rienstra W, van Raaij TM, ten Hagen AJ, Dijkstra B, Zijlstra WP, et al. Duloxetine in osteoarthritis (doa) study: study protocol of a pragmatic open-label randomised controlled trial assessing the effect of preoperative pain treatment on postoperative outcome after total hip or knee arthroplasty. BMJ Open. 2016;6(3):e010343. 14. Ho KY, Tay W, Yeo MC, Liu H, Yeo SJ, Chia SL, et al. Duloxetine reduces morphine requirements after knee replacement surgery. Br J Anaesth. 2010; 105(3):371–6. 15. YaDeau JT, Brummett CM, Mayman DJ, Lin Y, Goytizolo EA, Padgett DE, et al. Duloxetine and subacute pain after knee arthroplasty when added to
https://openalex.org/W2606539243	https://www.frontiersin.org/articles/10.3389/fpsyg.2017.00603/pdf	English	null	Planning and Performance in Small Groups: Collective Implementation Intentions Enhance Group Goal Striving	Frontiers in psychology	2,017	cc-by	10,680	ORIGINAL RESEARCH published: 19 April 2017 doi: 10.3389/fpsyg.2017.00603 Edited by: Martin S. Hagger, Curtin University, Australia Reviewed by: Chunqing Zhang, Hong Kong Baptist University, Hong Kong Carine Meslot, Université Grenoble Alpes, France Correspondence: J. Lukas Thürmer lukas.thuermer@uni-konstanz.de Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Received: 20 January 2017 Accepted: 03 April 2017 Published: 19 April 2017 Citation: Thürmer JL, Wieber F and Gollwitzer PM (2017) Planning and Performance in Small Groups: Collective Implementation Intentions Enhance Group Goal Striving. Front. Psychol. 8:603. doi: 10.3389/fpsyg.2017.00603 Planning and Performance in Small Groups: Collective Implementation Intentions Enhance Group Goal Striving J. Lukas Thürmer1,2,3, Frank Wieber3,4 and Peter M. Gollwitzer3,5 1 Department of Politics and Public Administration, University of Konstanz, Konstanz, Germany, 2 Department of Psychology and Learning Research and Development Center, University of Pittsburgh, Pittsburgh, PA, USA, 3 Department of Psychology, University of Konstanz, Konstanz, Germany, 4 Zurich University of Applied Sciences, School of Health Professions, Zurich, Switzerland, 5 Department of Psychology, New York University, New York, NY, USA There are two key motivators to perform well in a group: making a contribution that (a) is crucial for the group (indispensability) and that (b) the other group members recognize (identiﬁability). We argue that indispensability promotes setting collective (“We”) goals whereas identiﬁability induces individual (“I”) goals. Although both goals may enhance performance, they should align with different strategies. Whereas pursuing collective goals should involve more cooperation, pursuing individual goals should involve less cooperation. Two experiments support this reasoning and show that planning out collective goals with collective implementation intentions (cIIs or “We-plans”) relies on cooperation but planning out individual goals with individual implementation intentions (IIs or “I-plans”) does not. In Experiment 1, three-member groups ﬁrst formed a collective or an individual goal and then performed a ﬁrst round of a physical persistence task. Groups then either formed a respective implementation intention (cII or II) or a control plan and then performed a second round of the task. Although groups with cIIs and IIs performed better on a physical persistence task than respective control groups, only cII groups interacted more cooperatively during task performance. To conﬁrm the causal role of these interaction processes, Experiment 2 used the same persistence task and manipulated whether groups could communicate: When communication was hindered, groups with cIIs but not groups with IIs performed worse. Communication thus qualiﬁes as a process making cIIs effective. The present research offers a psychology of action account to small group performance. Keywords: cooperation and interaction, collective implementation intentions, small group performance, motivation, physical persistence Citation: Thürmer JL, Wieber F and Gollwitzer PM (2017) Planning and Performance in Small Groups: Collective Implementation Intentions Enhance Group Goal Striving. Front. Psychol. 8:603. doi: 10.3389/fpsyg.2017.00603 Indispensability versus Identiﬁability: Setting Collective versus Individual Goals What may be the actions and responses that group members choose to attain collective and individual goals during goal striving? Research on conﬂict resolution shows that positively interdependent goals lead to cooperative interaction, such as helping and talking to each other (Deutsch, 1949). In the context of conjunctive physical persistence, such cooperative interaction should surface in increased and more group focused verbal interaction, including encouragement, discussing the common goal, and how the group is doing (Deutsch, 2011). Given that collective goals highlight positive interdependence, group members should strive for them cooperatively exhibiting just this type of cooperative verbal interaction. Assuming that individual goals do not highlight positive interdependence, group members should strive for them less cooperatively. Testing this prediction thus not only requires setting collective goals (as commonly triggered by indispensability) versus individual goals (as commonly triggered by identiﬁability) but also supporting the respective goal striving route. Small group research has identiﬁed two primary motivators for group members to perform well: (a) one’s contribution is crucial for the group (indispensability) and (b) the other group members can recognize one’s contribution (identiﬁability) (Karau and Williams, 1993; Kerr and Hertel, 2011). Indispensability is motivating because one expects to make a crucial contribution to a valued group outcome or result (Kerr and Hertel, 2011). Such positive outcomes include attaining a group performance goal, receiving a group reward, or winning against another team. Because these outcomes all apply to the entire group, the goal matching this mechanism is best described as collective (e.g., “We want to break the record”). If the group attains the collective goal, all group members beneﬁt and one group member’s contributions beneﬁt all other group members as well. Therefore, collective goals triggered by indispensability have a positive interdependence within the group (Deutsch, 1949). Identiﬁability is motivating because a group member expects a positive outcome due to her own, individual contribution (Karau and Williams, 1993). Such positive outcomes include earning praise, receiving an individual reward for exceptional performance, or outperforming the other group members. 1Small group research has traditionally investigated indispensability and identiﬁability by manipulating task demands. In the present research, we focus on the individual’s own perspective that can emphasize one or the other process. INTRODUCTION Imagine lifting a heavy ball together with team mates in a small group. You realize that the other group members could not lift the ball without your help. You feel energized and try really hard because you know that your group needs you and that your contribution really makes a diﬀerence. Now imagine the same situation from a diﬀerent perspective: You are in your group and you realize April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org Collective Implementation Intentions Thürmer et al. Kerr et al., 2007). Thus, although individual goals are not necessarily competitive (cf. Van Lange, 1999; Murphy and Ackermann, 2014), they do not focus on the group outcome. In sum, then, collective as well as individual goals may motivate group members to perform well. In the present paper, we go one step further and analyze how group members act to attain these collective and individual goals. that the other group members can see your contribution. If you are the ﬁrst to give up holding the ball, everybody will know that it was you who failed; if you push through until somebody else gives up, everybody will notice that too. Again, you feel energized and try really hard because you want to do better than the other group members. These two perspectives1 reﬂect the two most commonly studied motivators in small groups (Karau and Williams, 1993; Kerr and Hertel, 2011): indispensability (your group needs you) and identiﬁability (the other group members can recognize your contribution). Goal Setting and Goal Striving: Setting Goals and Making Plans Indispensability leads group members to focus on outcomes for the entire group (i.e., we-goals), and accordingly group members use cooperative behaviors to attain these goals. In contrast, identiﬁability leads group members to focus on outcomes for oneself (i.e., I-goals), and accordingly group members may attain these goals without cooperation. Individuals attain and strive for their goals more successfully when they form additional if-then plans (implementation intentions, IIs). We argue that groups can use such if-then plans in two ways: support indispensability-related we-goals with new we-plans (collective implementation intentions, cIIs) or identiﬁability-related I-goals with traditional I-plans. Both types of plans should enhance performance, but only we-plans should increase cooperative group interaction. The psychology of action (Gollwitzer and Bargh, 1996; Gollwitzer and Moskowitz, 1996) distinguishes between a ﬁrst step of committing strongly to one’s goal (goal setting) and a second step where one has to implement goal-directed actions and responses—a process referred to as goal striving (Lewin, 1926; Lewin et al., 1944; Heckhausen and Gollwitzer, 1987; Gollwitzer, 1990). In support of the distinction between goal setting and goal striving, a meta-analysis (Webb and Sheeran, 2006) on experimental studies that manipulated the strength of the goal showed that a medium-to-large (Cohen, 1992) increase in commitment (d = 0.66) only led to a small-to-medium change in respective behavior (d = 0.36); an eﬀect largely due to people who are strongly committed but fail to act on their intention (Sheeran, 2002). Fully understanding intention- behavior relations thus requires analyzing goal setting as well as goal striving. Supporting Group Goal Striving: Forming Collective versus Individual Implementation Intentions individual goals, supporting individual as well as collective goal striving with respective if-then plans should therefore improve performance. However, while cIIs should rely on cooperation to improve performance, this does not have to be the case for IIs. Recent research shows that if-then planning also increases the rate of goal attainment in small group performance (Wieber et al., 2012, 2013; Thürmer et al., 2015a). For instance, if-then planning has been shown to help groups attain their goal of making informed decisions in hidden proﬁle situations (Thürmer et al., 2015b), to curb their investments in an escalation of commitment paradigm (Wieber et al., 2015), and to improve their performance in an interactive puzzle task (Wieber et al., 2017, unpublished). In some of these studies, groups used traditional IIs that refer to the individual (Wieber et al., 2017, unpublished); but in other studies, groups used new cIIs that refer to the group (Thürmer et al., 2015b; Wieber et al., 2015). Since all these studies consistently report that groups were more likely to attain their performance goal when forming additional if-then plans, group members seem to be able to use new cIIs as well as the traditional individual IIs to increase their performance. However, these reported studies did not compare the eﬀects of cIIs and IIs in a single design, and a systematic test comparing the eﬀects of individual versus cIIs as well as their underlying processes is still lacking. In two experiments, groups either set individual or collective goals and then performed two rounds of the persistence task (baseline and experimental). After the baseline round, groups furnished their goal with a respective plan to ignore muscle pain and tell themselves they can do it (i.e., a cII or an individual II). Such if-then plans to ignore negative aﬀect and to increase self-eﬃcacy feelings have been found to be highly eﬀective (e.g., Bayer and Gollwitzer, 2007; Schweiger Gallo et al., 2009; Thürmer et al., 2013). In Experiment 1, we further established two control conditions without respective if-then plans to assess whether forming additional if-then plans improves performance. We expected that both types of plans (i.e., cIIs and IIs) increase performance but lead to more versus less cooperative verbal interaction during task performance. In Experiment 2, we followed up on this assumed process and manipulated whether the task allowed for communication or hindered communication. Supporting Group Goal Striving: Forming Collective versus Individual Implementation Intentions Because we assumed that cIIs but not IIs rely on cooperative interaction to increase performance, we expected cIIs to lead to better performance when the task supported communication. IIs, on the other hand, should be eﬀective when communication is hindered. We thus propose that group members can eﬀectively regulate their goal striving by forming IIs that refer to the group (we, us, our). Like individual IIs, such cIIs are if-then plans that specify when, where, and how to act toward a set goal. Diﬀerent from IIs that refer the individual (I, me, mine) and support an individual goal, cIIs refer to the group (e.g., “And if we encounter situation S, then we will show response R!”) and support a collective goal. If increases in cooperation are indeed due to eﬀective goal striving (and not merely goal setting), supporting respective goal striving should magnify the diﬀerences between collective and individual goals. cIIs should activate the collective goal striving route and thus increase cooperation. Because IIs support the goals they are set for, referring to the group should therefore support cooperation. In contrast, an individual II may not activate cooperative strategies because it only refers to oneself. In sum, cIIs as well as IIs should increase group performance but cIIs should increase cooperation within the group. Indispensability versus Identiﬁability: Setting Collective versus Individual Goals Because these positive outcomes all apply to a single group member and not to the entire group, the goal matching this mechanism is best described as individual (e.g., “I want to win”; A simple way to support eﬃcient goal striving is planning out in advance when, where, and how to strive for a set goal in an if-then format (e.g., “And if I encounter situation S, then I will show the goal-directed response R!”; Gollwitzer, 1993, 1999, 2014). By forming such IIs, one commits to performing the speciﬁed behavior in the pre-planned situation. Thereby, one is more likely to act on and attain one’s goal (Gollwitzer and Sheeran, 2006; Adriaanse et al., 2011; Bélanger-Gravel et al., 2013; Toli et al., 2016). In the present paper, we suggest that diﬀerent types of IIs support striving for collective versus individual goals. April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org 2 Collective Implementation Intentions Thürmer et al. EXPERIMENT 1: DO COLLECTIVE IMPLEMENTATION INTENTIONS SUPPORT COOPERATIVE INTERACTION AND PERFORMANCE? The aims of Experiment 1 were twofold: First, we sought to establish that cIIs and individual IIs improve group performance in comparison to respective goals. Because indispensability (which should usually trigger collective goals) and identiﬁability (which should usually trigger individual goals) improve performance in such tasks, we expected that cIIs as well as individual IIs improve performance in comparison to the respective mere goals. Second, we sought to investigate if cIIs indeed lead to diﬀerent interaction patterns during goal striving than IIs. Positively interdependent goals commonly are associated with cooperative interaction, and supporting collective goals with cIIs should therefore intensify group interaction. In freely interacting groups, this intense group interaction should express itself in a high amount of verbal communication between group members. Related to this, the content of the verbal communication between group members should also reﬂect the respective type of goal striving. Research in psycholinguistics emphasizes that personal pronouns can be markers of one’s identity (Pennebaker et al., 2003) and research on social identity has shown that group-related pronouns can be indicative of group processes such as cooperation (Perdue et al., 1990; Brewer and Gardner, 1996). If cIIs support cooperative goal striving, group members with cIIs should therefore refer 2We used the formulas provided by Bliese (1998). Because of the poor agreement between group members, we additionally analyzed all scales assuming independence/without aggregating to the group level. This did not change the results. In Experiment 1, two items were excluded from the Klein et al. scale (“Quite frankly, I don’t care if I achieve this goal or not.” [reverse scored]/I think this is a good goal to shoot for”) because they reduced reliability (α < 0.50). The Present Research We analyzed whether planning out how to strive for collective and individual goals with respective cIIs and individual IIs increases performance and leads to more versus less cooperative interaction. We used a conjunctive physical persistence task where freely interacting groups were asked to hold a medicine ball as long as possible (Bray, 2004). We chose this task because each group member has to contribute equally and is therefore indispensable for performance; moreover, because group members are interacting face to face, it is easy to identify who failed ﬁrst (Kerr et al., 2007; Weber and Hertel, 2007; Kerr and Hertel, 2011). According to our earlier reasoning that indispensability triggers collective goals and that identiﬁability triggers April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org 3 Collective Implementation Intentions Thürmer et al. more to the group in their verbal communications by using ﬁrst person plural pronouns (we, us, our) and use more cooperative words. instructions individually, repeated their plans silently, envisioned them in their mind’s eye, and ﬁnally wrote them down. This procedure took about 5 min. p To measure the impact of this manipulation, a second, experimental round of the persistence task followed. To rule out the possibility that cIIs increase persistence because of increased goal commitment (a goal-setting variable), participants then responded to three goal commitment items (“It’s hard to take this goal seriously [reverse scored]/I am strongly committed to pursuing this goal/It wouldn’t take much to make me abandon this goal [reverse scored]” 1: not agree at all to 7: agree completely, Cronbach’s α = 0.71, ICC(1) = −0.12, ICC(2) = −0.47),2 adapted from Klein et al. (2001). Moreover, at the group level, increased group identiﬁcation might improve performance, which also does not qualify as a goal striving process. Participants thus responded to seven group identiﬁcation items (“I identify with my group/It is important to me to belong to my group/The fact that I belong to my group has little to do with how I see myself [reverse scored]/I am happy that I belong to my group/I often regret that I belong to my group [reverse scored]/I feel strong ties with my group/In general, I like belonging to my group” 1: disagree to 7: agree completely, Cronbach’s α = 0.84, ICC(1) = 0.26, ICC(2) = 0.51), adapted from Leach et al. (2008). Finally, participants provided demographic information including their height, were debriefed, thanked, and paid. Procedure After obtaining informed consent including a general ﬁtness check, the experimenter explained that the study investigated persistence in teams. Participants were to hold a ball simultaneously as long as possible by standing in a triangle and stretching out their dominant arm (Bray, 2004). For this task, groups were asked to form the goal “We (I) want to hold the ball as long as possible” (individual phrasing in parentheses) that was written on a board. Participants then performed the ﬁrst round of the task and the experimenter measured how long the group persisted. Next, participants received a paper-and-pencil form that included the manipulation of the referent and implementation intention factors. To test whether IIs improve persistence, experimental groups either added the collective if-then plan (cII) “And if our muscles hurt, then we will ignore the pain and tell ourselves: We can do it!” to their collective goal or the individual if-then plan (II) “And if my muscles hurt, then I will ignore the pain and tell myself: I can do it!” to their individual goal. The referent factor was thus manipulated by either referring to the group (we/collective) or to the individual (I). To make sure that individual and collective control groups had the same task-relevant knowledge, they were asked to add: “We (I) will ignore our (my) muscle pain and tell ourselves (myself): We (I) can do it!” (individual phrasing in parentheses). The content of these instructions therefore did not diﬀer between conditions apart from the if-then structure of the IIs. Participants read the Participants and Design One hundred and ﬁfty-six students from the University of Konstanz (117 females) with a mean age of 22.58 years (SD = 4.40) participated in return for coﬀee vouchers, 4€ (i.e., about 5$), or partial course credit. Participants were invited to the laboratory in same-sex triads (52 triads, 39 female) and a male experimenter randomly assigned groups to a 2 (Implementation Intention: yes vs. no) × 2 (Referent: individual vs. collective) factorial design. We used triads instead of dyads because some group researchers have argued that group phenomena might operate diﬀerently or not even occur in dyads (see Moreland, 2010; Williams, 2010, for a discussion). One participant in a cII group reported pain from a past injury (the trial was aborted immediately), one collective control group was not recorded because of hardware failure, two groups had members who were much older (3 SDs over the mean age of the sample; 1 collective control group, 1 II group), and one group stated during debrieﬁng that they had formed IIs although they were in the collective control condition (including these groups in the analysis did not change the pattern of results); 47 triads (35 female) remained for analyses. A power analysis (1 – β = 0.70) with G∗Power (Faul et al., 2007, 2009) showed that our sample size allows detecting a medium-to-large eﬀect (η2 p = 0.15) in our four-cell design. Dependent Measures We recorded how long groups held the medicine ball in seconds per trial. As common in research on group persistence (e.g., Kerr et al., 2012), the diﬀerence between the experimental and the baseline measure was computed to measure the impact of the planning manipulation on persistence. The audio recordings made during the trials were transcribed by a research assistant, and the word count function of the computer program AtlasTi (Muhr, 2012) counted the number of words per trial. We used the diﬀerence between the baseline and the experimental round to measure the impact of the planning manipulation on verbal communication. Two independent coders identiﬁed words representing group cooperation (e.g., teamwork, support, help; inter-coder agreement = 72%). In line with linguistic research on pronouns and identity (Pennebaker et al., 2003), we further coded the ﬁrst person plural pronouns (we, us, ours) in the cooperation category. Frontiers in Psychology \| www.frontiersin.org Results and Discussion Unless indicated otherwise, we analyzed the data with an ANOVA with Implementation Intention (yes vs. no) and Referent (individual vs. collective) as between factors. Equivalence of Conditions and Baseline Analysis All participants copied their respective goals and plans to the form correctly. Group identiﬁcation scores, M = 5.26, 3Entering gender as a covariate had a marginal main eﬀect, F(1,42) = 3.86, p = 0.06, η2 p = 0.08, but it did not change the observed referent eﬀect, F(1,42) = 9.44, p < 0.01, η2 p = 0.18, and the implementation intention eﬀect, F(1,42) = 5.68, p = 0.02, η2 p = 0.12. Equivalence of Conditions and Baseline Analysis All participants copied their respective goals and plans to the form correctly. Group identiﬁcation scores, M = 5.26, April 2017 \| Volume 8 \| Article 603 4 Collective Implementation Intentions Thürmer et al. other in the experimental round than groups in the individual conditions (Table 1), F(1,43) = 8.53, p = 0.01, η2 p = 0.17. However, this main eﬀect was qualiﬁed by an Implementation Intention × Referent interaction, F(1,43) = 5.06, p = 0.03, η2 p = 0.11. Groups in the control conditions did not diﬀer in the amount they spoke, F(1,43) = 0.25, p = 0.62, but the cII lead to more communication in comparison to the II, F(1,43) = 12.10, p < 0.01, η2 p = 0.22. Planned contrasts showed that the cII led to more communication than all other conditions, t(43) = 2.52, p = 0.02, and the II actually led to less communication than all other conditions, t(43) = 3.42, p < 0.01. Collective planning with the cII thus indeed lead to more verbal group interaction. SD = 0.96, and goal commitment scores, M = 5.99, SD = 0.78, were generally high and did not diﬀer between conditions, independent of whether the scores were aggregated across group members or treated as independent, Fs < 1, ps > 0.50. Increased motivation or stronger group identiﬁcation therefore do not qualify as alternative explanations for the expected performance improvements through if-then planning. Entering baseline persistence in a preliminary ANOVA surprisingly showed a marginal Implementation Intention × Referent interaction, F(1,43) = 3.87, p = 0.06, η2 p = 0.08. As the implementation intention factor was not manipulated until after this baseline measure, it is not plausible that the plan condition could have inﬂuenced persistence at that point. Instead, this eﬀect may reﬂect diﬀerent ability-levels of the groups. In line with this reasoning, the interaction became non-signiﬁcant when entering the mean height of the group members as a covariate, F(1,42) = 1.83, p = 0.18, η2 p = 0.04. Accordingly, we calculated the diﬀerence between experimental and baseline measures (Round 2 – Round 1) to assess the impact of the manipulation; including height as a covariate did not change the following persistence analysis. Dependent Variable: Persistence In line with previous research (e.g., Lount et al., 2008), groups deteriorated from baseline to experimental round (Table 1). To test whether forming if-then plans improved persistence, we entered the persistence score (experimental minus baseline) into the ANOVA. As expected, groups with an implementation intention (cII or II) persisted relatively longer in the experimental round than groups with a control plan, F(1,43) = 5.11, p = 0.03, η2 p = 0.11 (Figure 1). This supports our prediction that an II as well as a cII to ignore muscle pain and to tell oneself that one can do well on the task improves persistence. Moreover, a main eﬀect of referent occurred, F(1,43) = 11.16, p < 0.01, η2 p = 0.21: Groups that had made collective plans persisted relatively longer than groups with individual plans. The main eﬀects were not qualiﬁed by an Implementation Intention × Referent interaction, F(1,43) = 0.63, p = 0.43.3 In sum, the observed results are in line with the idea that if-then planning supports group performance. But do the two types of implementation intentions (cIIs and IIs) rely on diﬀerent processes? In sum, Experiment 1 shows that if-then planning improves group performance, and that cIIs and IIs lead to diﬀerent group interaction patterns. While cIIs left verbal communication between group members intact and led group members to speak cooperatively with each other, IIs lead to less verbal communication. This pattern supports the assumption that group members can strive for goals collectively or individually, and that forming respective if-then plans supports the matching type of goal striving (collective or individual). We conducted Experiment 2 to conﬁrm the causal impact of this assumed process by either supporting or hindering cooperative verbal interaction. Equivalence of Conditions and Baseline Analysis No main or interaction eﬀects for the number of words spoken, Fs(1,43) < 1.90, ps > 0.17, or the number of cooperative words spoken, Fs(1,43) < 2.90, ps > 0.10, were observed at baseline, and we therefore also calculated diﬀerence scores. We also assumed that groups with cIIs should communicate more cooperatively than groups with IIs. To test this assumption, we entered the cooperation score into the ANOVA. This ANOVA showed a Referent main eﬀect, F(1,43) = 7.41, p = 0.01, η2 p = 0.15, that was qualiﬁed by a marginal Referent × Implementation Intention interaction, F(1,43) = 3.66, p = 0.06, η2 p = 0.08. Pairwise comparisons showed that groups with cIIs spoke more cooperatively with each other than groups with IIs, F(1,43) = 10.60, p < 0.01, η2 p = 0.20; no Referent eﬀect occurred for control groups, F(1,43) = 0.63, p = 0.43. One may argue that this eﬀect is mainly driven by priming the collective referent “we;” however, even within the collective referent conditions that both referred to “we,” if-then planning with cIIs tended to increase cooperative communication, F(1,43) = 3.42, p = 0.07, η2 p = 0.07. This overall pattern of results is thus in line with our assumption that cIIs but not IIs support cooperative collective goal striving. Process Measure: Group Interaction We argued that cIIs support the use of cooperative task strategies in comparison to IIs. We thus expected that collective if-then plans would lead to more communication than individual if-then plans. To test this prediction, we entered the word count diﬀerence score (experimental minus baseline) into the ANOVA. Indeed, groups in the collective conditions spoke more to each The aim of Experiment 2 was to directly test whether cIIs improve performance via cooperative group interaction. To test this process hypothesis, we opted to manipulate the assumed process variable (group interaction), as recommended by Spencer et al. (2005; see also Bullock et al., 2010). Our reasoning was as follows: If cIIs rely more on group interaction than IIs, hindering group interaction should impair performance in cII groups but not in II groups. To this end, groups were either encouraged to April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org 5 Collective Implementation Intentions Thürmer et al. TABLE 1 \| Persistence, verbal communication, and communication content measures by Implementation Intention (II) and Referent (Experiment 1). Measure vs. collective) × 2 (Communication: supported vs. hindered) between factorial design. Three groups (1 in the II condition and 2 in the cII condition) did not follow task instructions and communicated despite being prompted not to (their values were in the 95% CI range of the communication-supported condition); thirty-eight triads (27 females) remained for statistical analysis. A power analysis (1 −β = 0.70) with G∗Power (Faul et al., 2007, 2009) indicates that our sample size allows detecting an eﬀect of the size observed in Experiment 1 (η2 p = 0.15) in our four-cell design. communicate with each other while performing the persistence task or were prevented from communicating. Moreover, groups formed either cIIs (collective referent) or IIs (individual referent) between the ﬁrst and the second round. Assuming that cIIs achieved their eﬀects by enhancing cooperative interaction between group members, we hypothesized that cIIs should lead to better performance when the task supported communication between group members but that this should not be true for IIs. Frontiers in Psychology \| www.frontiersin.org Process Measure: Group Interaction Referent Individual Collective Measure II: no II: yes cII: no cII: yes Seconds holding the ball Persistence Baseline 190.86 (54.14) [159.60; 222.11] 212.60 (81.33) [154.42; 270.78] 193.58 (77.49) [144.35; 242.82] 139.82 (44.54) [109.89; 169.74] Experimental 142.07 (44.20) [116.55; 167.59] 181.80 (61.09) [138.10; 225.50] 176.00 (73.39) [129.37; 222.63] 159.64 (41.58) [131.70; 187.57] Difference (dependent measure) −48.79 (28.75) [−65.39; −32.19] −30.80 (62.03) [−75.17; 13.57] −17.58 (39.53) [−42.70; 7.53] 19.82 (34.76) [−3.54; 43.17] Number of words spoken Verbal communication Baseline 96.57 (86.62) [46.56; 146.59] 174.40 (135.78) [77.27; 271.53] 104.08 (94.44) [44.08; 164.08] 100.73 (84.81) [43.75; 157.70] Experimental 52.14 (60.81) [17.03; 87.25] 71.80 (104.68) [−3.08; 146.68] 72.75 (76.82) [23.94; 121.56] 99.00 (91.02) [37.85; 160.15] Difference (dependent measure) −44.43 (56.94) [−77.30; −11.56] −102.60 (93.28) [−169.33; −35.87] −31.33 (67.93) [−74.49; 11.83] −1.73 (42.69) [−30.40; 26.95] Number of cooperative words spoken Communication content Baseline 3.21 (2.86) [1.56; 4.87] 5.40 (4.84) [1.94; 8.86] 3.75 (3.44) [1.56; 5.94] 2.64 (1.80) [1.42; 3.85] Pronouns 2.86 (2.51) [1.41; 4.30] 4.70 (4.40) [1.55; 7.85] 3.42 (3.40) [1.26; 5.57] 1.91 (1.38) [0.99; 2.83] Other 0.36 (0.84) [−0.13; 0.84] 0.70 (0.90) [0.02; 1.38] 0.33 (0.65) [−0.08; 0.75] 0.73 (0.90) [0.12; 1.33] Experimental 1.07 (1.86) [0.00; 2.14] 2.10 (4.33) [−1.00; 5.20] 2.58 (2.87) [0.76; 4.41] 4.00 (4.75) [0.81; 7.19] Pronouns 1.07 (1.86) [0.00; 2.14] 1.90 (4.01) [−0.97; 4.77] 2.50 (2.84) [0.69; 4.31] 3.73 (4.45) [0.74; 6.72] Other 0.00 (n/a) [n/a] 0.20 (0.42) [−0.10; 0.50] 0.08 (0.29) [−0.10; 0.27] 0.27 (0.47) [−0.04; 0.59] Combined difference (dependent measure) −2.14 (2.28) [−3.46; −0.83] −3.30 (4.50) [−6.52; −0.08] −1.17 (2.48) [−2.74; 0.41] 1.36 (3.80) [−1.19; 3.92] n 14 triads 10 triads 12 triads 11 triads Standard deviations are in parentheses and 95% conﬁdence intervals of the condition mean are in brackets. TABLE 1 \| Persistence, verbal communication, and communication content measures by Implementation Intention (II) and Referent (Experiment 1). TABLE 1 \| Persistence, verbal communication, and communication content measures by Implementation Intention (II) and Referent (Experiment 1). Persistence and Communication support communication, groups were told that they are allowed to talk to each other, that they should face each other, and wear the headset around their necks. Audio recordings were made as a manipulation check. To check whether the Communication factor manipulation was successful, we entered a word count of the ﬁrst trial into the ANOVA: Groups in the communication-supported condition indeed spoke more than groups in the communication- hindered condition, F(1,34) = 25.67, p < 0.01, η2 p = 0.43 (Table 2). Even though groups in the “communication hindered” conditions did not manage to remain completely silent, the very large eﬀect size (η2 p = 0.43 is equivalent to d = 1.68) suggests that our manipulation was successful. As expected, we neither observed an Implementation Intention Referent main eﬀect, F(1,34) = 0.92, p = 0.34, η2 p = 0.03, nor an Implementation Intention Referent × Communication interaction, F(1,34) = 0.45, p = 0.51, η2 p = 0.01, at this point before the plan manipulation. p After forming the individual versus collective goals with IIs used in Experiment 1, groups performed the second round of the task. Participants then responded to the questionnaires assessing goal commitment [Cronbach’s α = 0.74, ICC(1) = −0.00, ICC(2) = −0.00]2 and group identiﬁcation [Cronbach’s α = 0.91, ICC(1) = 0.13, ICC(2) = 0.32] used in Experiment 1. To check whether cII groups wanted to comply with their plan as much as II groups, participants also responded to a three- item questionnaire measuring plan commitment [“It is important for me to fulﬁll my plan/It would be a shame if I could not fulﬁll my plan/I feel committed to my plan,” 1: not at all to 5: very much, Cronbach’s α = 0.80, ICC(1) = 0.10, ICC(2) = 0.25] adapted from Wieber et al. (2015). Lastly, we asked participants for demographic information, including their major and semester of study. To test whether the communication manipulation alone impacted performance, the ﬁrst round persistence measure was entered into the ANOVA. Groups in the communication-supported condition outperformed groups in the communication-hindered condition (Table 2), F(1,34) = 7.48, p = 0.01, η2 p = 0.18. This suggests that intense interaction increases performance in our physical persistence task, which is consistent with the Referent main eﬀect observed in Experiment 1. Results and Discussion Unless indicated otherwise, we analyzed the data with an ANOVA with Implementation Intention Referent (collective: cII vs. individual: II) and Communication (supported vs. hindered) as between factors. Equivalence of Conditions All participants copied their respective goals and plans to the form correctly. Goal commitment, M = 5.10, SD = 0.78, plan commitment, M = 4.02, SD = 0.75, and group identiﬁcation, M = 5.33, SD = 1.11, were high and did not diﬀer between conditions, Fs < 2.20, ps > 0.14. Participants across conditions thus equally wanted to comply with the adopted goals and plans, and cared about their group. Persistence and Communication As expected, we neither observed an Implementation Intention Referent main eﬀect, F(1,34) = 0.37, p = 0.55, η2 p = 0.01, nor an Implementation Intention Referent × Communication interaction, F(1,34) = 2.32, p = 0.14, η2 p = 0.06, at this point before the plan manipulation. Participants and Design Experiment 2 followed the same procedure as Experiment 1, with the following diﬀerence: Before the ﬁrst round of the task, instructions were varied to manipulate the Communication factor. To hinder communication, groups were instructed not to talk to each other, to each look at a separately marked point on the wall away from the group, and to wear a headset over their ears; to One hundred and twenty-three university students (90 females) with a mean age of 22.13 years (SD = 2.85) participated in return for 4€ or partial course credit. Participants were invited to the laboratory in same-sex triads (41 triads, 30 females) and a female experimenter randomly assigned them to one of four conditions in a 2 (Implementation Intention Referent: individual April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org 6 Thürmer et al. Collective Implementation Intentions FIGURE 1 \| Mean persistence scores (experimental [s] minus baseline [s]) by Implementation Intention and Referent (Experiment 1). Error bars represent standard errors. II: Individual implementation intention; cII: collective implementation intention. FIGURE 1 \| Mean persistence scores (experimental [s] minus baseline [s]) by Implementation Intention and Referent (Experiment 1). Error bars represent standard errors. II: Individual implementation intention; cII: collective implementation intention. Persistence and If-then Planning We next tested how collective and individual if-then planning impacted performance and thus again calculated the persistence score (experimental minus baseline). Because we were interested April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org 7 Thürmer et al. Collective Implementation Intentions TABLE 2 \| Persistence measures by Implementation Intention Referent and Task Communication (Experiment 2). Implementation Intention Referent Individual (II) Collective (cII) Measure Communication hindered Communication supported Communication hindered Communication supported Manipulation check: number of words spoken Baseline 1.43 (3.37) [−0.98; 3.84] 21.96 (12.33) [12.49; 31.44] 2.78 (5.69) [−1.60; 7.15] 29.60 (24.62) [11.99; 47.21] Experimental 0.00 (n/a) [n/a] 12.78 (10.42) [4.77; 20.79] 0.00 (n/a) [n/a] 17.77 (13.07) [8.42; 27.11] Persistence Baseline (s) 118.90 (43.24) [87.97; 149.83] 190.11 (60.43) [143.66; 236.57] 134.22 (57.63) [89.93; 178.52] 154.50 (44.17) [122.90; 186.10] Experimental (s) 120.00 (61.10) [76.29; 163.71] 139.22 (37.47) [110.42; 168.02] 97.22 (28.28) [75.49; 118.96] 132.20 (51.37) [95.45; 168.95] Difference 1.10 (42.37) [−29.21; 31.41] −50.89 (48.60) [−88.25; −13.53] −37.00 (34.97) [−63.88; −10.12] −22.30 (25.86) [−40.80; −3.80] Difference z-transformed per communication condition (dependent measure) 1.84 (4.32) [−1.25; 4.93] −1.64 (5.30) [−5.72; 2.43] −2.05 (3.57) [−4.79; 0.70] 1.48 (2.82) [−0.54; 3.49] n 10 triads 9 triads 9 triads 10 triads Standard deviations are in parentheses and 95% conﬁdence intervals of the condition mean are in brackets. FIGURE 2 \| z-transformed persistence scores by Implementation Intention Referent and Communication (Experiment 2). Error bars represent standard errors. II: Individual implementation intention, cII: collective implementation intention. in the additional eﬀects of planning, we sought to account for the systematic baseline diﬀerences caused by the Communication factor. We therefore pooled the persistence diﬀerence scores per C i i di i (i ll d I l i Referent × Communication interaction, F(1,34) = 6.98, p = 0.01, η2 p = 0.17: As predicted, cII groups marginally performed better when communication was supported (Figure 2 d T bl 2) F(1 34) 3 53 0 07 2 0 09 4 Collective Implementation Intentions Thürmer et al. TABLE 2 \| Persistence measures by Implementation Intention Referent and Task Communication (Experiment 2). Persistence and If-then Planning Implementation Intention Referent Individual (II) Collective (cII) Measure Communication hindered Communication supported Communication hindered Communication supported Manipulation check: number of words spoken Baseline 1.43 (3.37) [−0.98; 3.84] 21.96 (12.33) [12.49; 31.44] 2.78 (5.69) [−1.60; 7.15] 29.60 (24.62) [11.99; 47.21] Experimental 0.00 (n/a) [n/a] 12.78 (10.42) [4.77; 20.79] 0.00 (n/a) [n/a] 17.77 (13.07) [8.42; 27.11] Persistence Baseline (s) 118.90 (43.24) [87.97; 149.83] 190.11 (60.43) [143.66; 236.57] 134.22 (57.63) [89.93; 178.52] 154.50 (44.17) [122.90; 186.10] Experimental (s) 120.00 (61.10) [76.29; 163.71] 139.22 (37.47) [110.42; 168.02] 97.22 (28.28) [75.49; 118.96] 132.20 (51.37) [95.45; 168.95] Difference 1.10 (42.37) [−29.21; 31.41] −50.89 (48.60) [−88.25; −13.53] −37.00 (34.97) [−63.88; −10.12] −22.30 (25.86) [−40.80; −3.80] Difference z-transformed per communication condition (dependent measure) 1.84 (4.32) [−1.25; 4.93] −1.64 (5.30) [−5.72; 2.43] −2.05 (3.57) [−4.79; 0.70] 1.48 (2.82) [−0.54; 3.49] n 10 triads 9 triads 9 triads 10 triads Standard deviations are in parentheses and 95% conﬁdence intervals of the condition mean are in brackets. TABLE 2 \| Persistence measures by Implementation Intention Referent and Task Communication (Experiment 2). FIGURE 2 \| z-transformed persistence scores by Implementation Intention Referent and Communication (Experiment 2). Error bars represent standard errors. II: Individual implementation intention, cII: collective implementation intention. FIGURE 2 \| z-transformed persistence scores by Implementation Intention Referent and Communication (Experiment 2). Error bars represent standard errors. II: Individual implementation intention, cII: collective implementation intention. FIGURE 2 \| z-transformed persistence scores by Implementation Intention Referent and Communication (Experiment 2). Error bars represent standard errors. II: Individual implementation intention, cII: collective implementation intention. Referent × Communication interaction, F(1,34) = 6.98, p = 0.01, η2 p = 0.17: As predicted, cII groups marginally performed better when communication was supported (Figure 2 and Table 2), F(1,34) = 3.53, p = 0.07, η2 p = 0.09.4 Referent × Communication interaction, F(1,34) = 6.98, p = 0.01, η2 p = 0.17: As predicted, cII groups marginally performed better when communication was supported (Figure 2 and Table 2), F(1,34) = 3.53, p = 0.07, η2 p = 0.09.4 in the additional eﬀects of planning, we sought to account for the systematic baseline diﬀerences caused by the Communication factor. We therefore pooled the persistence diﬀerence scores per Communication condition (i.e., collapsed across Implementation Intention Referent conditions) and then computed the respective z-scores (Table 2). 4Entering gender as a covariate into the model did not have an eﬀect, F(1,33) = 1.54, p = 0.22, and did not change the observed Implementation Intention Referent × Communication interaction, F(1,33) = 7.25, p = 0.01, η2 p = 0.18. As a proxy of familiarity, we coded how many group members had the same major and also were in the same semester (i.e., 1 = all group members are in a diﬀerent semester or have a diﬀerent major, 2 = two of the members are in the same semester and also have the same major, 3 = all group members have the same major and are in the same semester). Entering this variable as a covariate into the model neither had a main eﬀect, F(1,33) = 0.43, p = 0.52, η2 p = 0.01, nor did it change the observed interaction, F(1,33) = 6.94, p = 0.01, η2 p = 0.17. If-then Planning and Communication We also checked whether if-then planning changed verbal interaction in the communication condition. Entering the communication diﬀerence score (experimental minus baseline) into the model showed no main or interaction eﬀects, all Fs(1,34) < 2.76, all ps > 0.10; also no eﬀects evinced when entering the cooperation score into the model, all Fs(1,34) < 1, all ps > 0.65. Thus, cIIs did not further increase cooperative group interaction in Experiment 2. One reason for this ﬁnding may be that the groups who were instructed to communicate perceived the task as being highly cooperative and thus already interacted intensely during baseline. Future research should test this hypothesis. Implications for Small Group Performance Experiment 2 investigated whether group communication indeed qualiﬁes as a process causing the positive eﬀects of cIIs on group performance. We tested this assumption by manipulating whether the task at hand supported or hindered group communication. We found that cII eﬀects were greater when communication was supported, whereas individual II eﬀects were greater when communication was hindered. Together with Experiment 1 demonstrating that cIIs support cooperative verbal interaction, Experiment 2 suggests that cooperative group interaction does qualify as a process variable for cII eﬀects but not II eﬀects. Accordingly, it seems justiﬁed to distinguish between cIIs and individual IIs. Observing that cIIs improve persistence via communication is in line with recent research showing that social support can lead to group motivation gains (Hüﬀmeier et al., 2014). But our research also demonstrates that groups may perform eﬀectively without interacting (i.e., by striving individually), as is commonly found in the management literature (Lount and Wilk, 2014). Our research thus suggests that individual goals and plans may lead groups to perform well in conjunctive tasks, although not to the level of collective goals and plans. It is important to note that group members’ personality attributes may moderate our eﬀects. Implementation intention research shows that highly conscientious individuals do not beneﬁt much from receiving additional implementation intention instructions, supposedly because they already plan spontaneously (Webb et al., 2007). Analogously, the conscientiousness of the average member (cf. Neuman et al., 1999) might moderate cII eﬀects. Future research should test this assumption. Persistence and If-then Planning Entering this score as dependent variable into the ANOVA revealed the expected Implementation Intention 4Entering gender as a covariate into the model did not have an eﬀect, F(1,33) = 1.54, p = 0.22, and did not change the observed Implementation Frontiers in Psychology \| www.frontiersin.org April 2017 \| Volume 8 \| Article 603 8 Collective Implementation Intentions Thürmer et al. Thus, hindering communication impaired performance in cII groups. less cooperation. Supporting striving for collective goals with cIIs should therefore enhance group performance via intensifying cooperative interaction. Moreover, II groups marginally performed better when the communication was hindered, F(1,34) = 3.45, p = 0.07, η2 p = 0.09. This may suggest that communication without the focus on interdependence that collective goals and plans provide may be distracting instead of helpful. Future research should investigate this hypothesis. Two experiments support this hypothesis and show that cIIs improve group performance in a conjunctive physical persistence task via cooperative verbal communication. The beneﬁcial eﬀects of cIIs on group performance rely on improved goal striving, as II eﬀects on individual goal attainment: We observed performance improvements through cIIs when control groups received almost identical goals (same strategy with the same referent) only lacking the if-then format that is typical of IIs (Experiment 1). Moreover, the observed performance improvements by cIIs were not due to heightening participants’ goal commitment, but cIIs did increase cooperative group interaction. We manipulated the intensity of group interaction in Experiment 2 to conﬁrm the causal role of this assumed process: Groups with cIIs but not groups with IIs performed worse when the task hindered communication. Apparently, enhanced cooperative interaction qualiﬁes as a process associated with cII but not II eﬀects. GENERAL DISCUSSION Small group research has consistently identiﬁed two main motivators to work hard in a group: your group needs you (indispensability; Kerr and Hertel, 2011) and the other group members can recognize your contribution (identiﬁability; Karau and Williams, 1993). We argued that indispensability triggers collective goals (e.g., we want to beat our record), whereas identiﬁability triggers individual goals (e.g., I want to beat my record). The psychology of action highlights that, in addition to setting goals, people also need to eﬀectively implement goal- directed actions to secure goal attainment. Accordingly, we argued that group members should strive for collective goals by cooperating with each other but strive for individual goals with Our approach to motivation in groups may remind the reader of the work on goal setting and group performance (O’Leary- Kelly et al., 1994; Kleingeld et al., 2011). However, although forming IIs and setting challenging-and-speciﬁc goals both add speciﬁcity to one’s goal, these kinds of speciﬁcity diﬀer: In goal setting, one quantiﬁes the desired outcome (i.e., one speciﬁes a certain goal standard), which makes discrepancies between the actual state and the desired end state easier to detect. In contrast, IIs specify how to attain an already set goal in terms of when, where, and how to act toward it. Despite these diﬀerences between IIs and goal setting, the individual-collective distinction is crucial for both: Collective goals (Locke and Latham, 1990; Crown and Rosse, 1995; Crown, 2007) as well as cIIs improve performance by improving group interaction. Groups can thus actively regulate the interaction between group members. This April 2017 \| Volume 8 \| Article 603 Frontiers in Psychology \| www.frontiersin.org 9 Collective Implementation Intentions Thürmer et al. growing body of research is in line with the idea that groups are intentional entities that can regulate their behavior. group and still pursue goals individually. Lastly, others have also distinguished collective and individual motivation (e.g., Latham and Locke, 2007; Maciejovsky and Budescu, 2007, 2013; De Dreu et al., 2008; Levine and Smith, 2013; Maciejovsky et al., 2013). However, all these accounts have not distinguished between the incentive (goal) and the strategy to attain it (goal striving). In the light of modest intention-action relations (Sheeran, 2002; Webb and Sheeran, 2006), introducing this distinction is a crucial contribution of the present research. growing body of research is in line with the idea that groups are intentional entities that can regulate their behavior. Implications for the Psychology of Collective Action Traditionally, the psychology of action and implementation intention research have focused on individuals (Gollwitzer and Sheeran, 2006; Gollwitzer and Oettingen, 2011), and research on implementation intention eﬀects in groups is fairly recent (Wieber et al., 2012, 2013; Thürmer et al., 2015a). In the present research, we systematically investigated the implementation intention referent (i.e., We vs. I) and found that small groups can improve their performance by forming if-then plans that refer to the group (i.e., “we” if-then plans or cIIs). If-then planning is therefore not only eﬀective in individuals and with an individual referent but also in groups and with a collective referent. In sum, the present research demonstrates that planning out collective and individual goals improves group performance via two diﬀerent routes: Furnishing collective goals with cIIs increases cooperative interaction but furnishing individual goals with IIs does not. In this way, goal striving in groups with IIs helps groups perform to their full potential. Recent accounts have discussed small group self-regulation without pointing to if-then planning. First, small group approaches address how groups attempt to regulate their members’ behavior (e.g., through assigning roles and enforcing norms) and how the group members react (e.g., by capitulating or resisting; Peterson and Behfar, 2005; Levine et al., 2010). Our perspective is complementary to this view: To use cIIs, group members may have to accept pertinent roles and norms because they otherwise lack commitment to collective goals. Second, the group based self-regulation account (Sassenberg and Woltin, 2008; Jonas et al., 2010; Woltin and Sassenberg, 2015) assumes that by identifying as a group member, one self-regulates in the service of a group. The self-regulation processes are assumed to be the same as those at the individual level. Intra- individual processes, such as committing to a goal and a plan, are also crucial for implementation intention eﬀects. However, our distinction between cIIs and IIs is based on the referent (We vs. I) instead of the identiﬁcation. In line with our perspective, cIIs increased cooperative communication but did not change group identiﬁcation. A group member may thus identify with the AUTHOR CONTRIBUTIONS JLT, FW, and PG jointly designed the studies. JLT and FW supervised data collection and analyzed the data. JLT prepared a ﬁrst draft and JLT, FW, and PG jointly revised the manuscript. This research is part of JLT’s doctoral dissertation. JLT, FW, and PG jointly designed the studies. JLT and FW supervised data collection and analyzed the data. JLT prepared a ﬁrst draft and JLT, FW, and PG jointly revised the manuscript. This research is part of JLT’s doctoral dissertation. ETHICS STATEMENT This study was carried out in accordance with the recommendations of ethics committee of the University of Konstanz. All subjects gave written informed consent in accordance with the Declaration of Helsinki. FUNDING This work was supported by the German Research Foundation [DFG, GO 387/ 14-3]. REFERENCES J., and Williams, K. D. (1993). Social loaﬁng: a meta-analytic review and theoretical integration. J. Pers. Soc. Psychol. 65, 681–706. doi: 10.1037/0022- 3514.65.4.681 Pennebaker, J. W., Mehl, M. R., and Niederhoﬀer, K. G. (2003). Psychological aspects of natural language use: our words, our selves. Annu. Rev. Psychol. 54, 547–577. doi: 10.1146/annurev.psych.54.101601.145041 Kerr, N. L., Feltz, D. L., and Irwin, B. C. (2012). 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https://openalex.org/W4240780168	https://backend.orbit.dtu.dk/ws/files/161702613/cctc.201700811.pdf	English	null	Automated Determination of Oxygen‐Dependent Enzyme Kinetics in a Tube‐in‐Tube Flow Reactor	ChemCatChem	2,017	cc-by	5,029	Citation (APA): Ringborg, R. H., Pedersen, A. T., & Woodley, J. (2017). Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor. 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General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor Ringborg, Rolf Hoffmeyer; Pedersen, Asbjørn Toftgaard; Woodley, John Document Version Publisher's PDF, also known as Version of record General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a Selective oxidation is one of the most important transforma- tions in synthetic organic chemistry.[1–3] The necessity of achiev- ing high reaction yield in such transformations makes enzymes particularly interesting as potential catalysts, on account of their exquisite selectivity in comparison with their chemo-catalytic counterparts. However, for process application it is often diffi- cult to reach the required reaction intensity (reaction rate and product concentration). In particular, issues such as low enzy- matic activity, product/substrate inhibition, co-factor regenera- tion and unfavorable thermodynamic equilibria need to be solved using biocatalytic reaction engineering. These problems are commonly investigated by studying the kinetic behavior of an enzyme under different conditions. Subsequently, using these data, the challenges in reaching the required productivity can be addressed either by protein engineering or, alternatively, process engineering to circumvent kinetic limitations. However, it would be much more effective if solutions arose from a com- Selective oxidation is one of the most important transforma- tions in synthetic organic chemistry.[1–3] The necessity of achiev- ing high reaction yield in such transformations makes enzymes particularly interesting as potential catalysts, on account of their exquisite selectivity in comparison with their chemo-catalytic counterparts. However, for process application it is often diffi- cult to reach the required reaction intensity (reaction rate and product concentration). In particular, issues such as low enzy- matic activity, product/substrate inhibition, co-factor regenera- tion and unfavorable thermodynamic equilibria need to be solved using biocatalytic reaction engineering. These problems are commonly investigated by studying the kinetic behavior of an enzyme under different conditions. Subsequently, using these data, the challenges in reaching the required productivity can be addressed either by protein engineering or, alternatively, process engineering to circumvent kinetic limitations. However, it would be much more effective if solutions arose from a com- g yg p y On studying enzyme kinetics, it is important to measure ini- tial rates at substrate concentrations well above, as well as below, the true Michaelis constant(s), to determine kinetic pa- rameters with sufficient accuracy. In the study of oxygen-de- pendent enzymes, such investigations are notoriously difficult as a result of the limited solubility of oxygen in water, and to some extent, of the concomitant limited supply rate of oxygen. The challenge of controlling the oxygen concentration leads in many cases to conducting experiments at a single oxygen concentration (usually that in water, in equilibrium with air, at 276 mm). Link back to DTU Orbit Link back to DTU Orbit Citation (APA): Ringborg, R. H., Pedersen, A. T., & Woodley, J. (2017). Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor. ChemCatChem, 9(17), 3285 – 3288. https://doi.org/10.1002/cctc.201700811 this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim. Communications DOI: 10.1002/cctc.201700811 DOI: 10.1002/cctc.201700811 Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a bination of both approaches. Regardless of the approach taken, enzyme improvement naturally starts in the hands of the pro- tein engineer who typically screens for improved enzymes using single point measurements (i.e. at a single substrate con- centration) to go through many enzyme variants.[4] In this way, protein engineering is able to deliver improved enzymes, also catalyzing the conversion of non-natural substrates.[5] However, single point measurements can only reveal apparent kinetic constants, such as the so-called specificity constant (Vmax/KM), which can be misleading as the basis for selecting the optimal enzyme.[6–8] At points in development at which selection is made from a smaller pool of protein variants, it would be highly desirable to comprehensively quantify the kinetics, to have an adequate basis for deciding on the best enzyme for a given reaction, and reactor configuration. Likewise, it is necessa- ry to determine the activity of an enzyme of interest over the full range of potential operating conditions to be able to truly assess the possibilities for process implementation. On this premise, we suggest that comprehensive kinetic investigations should be integrated into the improvement cycle of an enzyme for application. In this way it would be possible to direct screen- ing to focus on evolving improved enzymatic kinetic properties, which are ideal for process implementation. To realize such a scheme, it is necessary to develop an automated characteriza- tion system.[9] Herein, we present one such system focused on collecting kinetic data for oxygen-dependent enzymes. Enzyme-mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such sys- tems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and lim- ited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube-in-tube reactor, operated in the low-dispersed flow regime to generate time-series data, with minimal material consumption. Experi- mental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data ob- tained, but also the necessity of making measurements in this way to enable the accurate evaluation of high KMO enzyme sys- tems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle. T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 85 ChemCatChem 2017, 9, 3285 – 3288 Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a Air saturation is however insufficient to achieve enzyme saturation for several industrially interesting oxidases[10–12] and, in any case, it introduces uncertainty into parameter estimations. Indeed, conventional experiments can only reveal apparent Michaelis constants which are confined to the tested parameter space and should therefore be compared with great care. Likewise, oxygen supply is often carried out by bubbling air through the reaction solution. However, in doing so, it is necessary to consider the stripping of any volatile sub- strate(s) and product(s), as well as potential enzyme deactiva- tion at the gas-liquid interface.[13] The constraint on the limited dissolved oxygen concentration in water can be alleviated by pressurizing the reactor or by using enriched air (to increase [a] Dr. R. H. Ringborg,+ A. Toftgaard Pedersen,+ Prof. J. M. Woodley Department of Chemical and Biochemical Engineering Technical University of Denmark DK-2800 Kgs. Lyngby (Denmark) E-mail: jw@kt.dtu.dk [b] Dr. R. H. Ringborg+ EchoSkye DK-2300 Copenhagen S (Denmark) [++] These authors contributed equally to this work. Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/ cctc.201700811. T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. 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. [a] Dr. R. H. Ringborg,+ A. Toftgaard Pedersen,+ Prof. J. M. Woodley Department of Chemical and Biochemical Engineering Technical University of Denmark DK-2800 Kgs. Lyngby (Denmark) E-mail: jw@kt.dtu.dk [b] Dr. R. H. Ringborg+ EchoSkye DK-2300 Copenhagen S (Denmark) [++] These authors contributed equally to this work. Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/ cctc.201700811. T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. 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. T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. 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. T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a We reasoned this would make the TiTR ideal for studying the kinetics of oxygen dependent bio- catalytic reactions, since the challenges of conventional sys- tems can be avoided by creating a bubble-free aeration system. The small dimensions of the inner tube (I.D/O.D. 230/ 410 mm) maximize the surface-to-volume ratio, which com- bined with the high oxygen permeability of Teflon AF-2400, enables very high oxygen supply rates. This TiTR allows opera- tion at dissolved concentrations of oxygen very close to the equilibrium value between the gas phase and the reaction medium, despite a low driving force (i.e. the reactor will oper- ate at a dissolved oxygen concentration within 99% of satura- tion). Additionally, by pressurizing both the inner and outer tube, the oxygen solubility in the reaction mixture can be in- creased proportionally. The setup therefore allows control over oxygen as a substrate in oxygen-dependent enzyme reactions. Furthermore, the TiTR satisfies the requirement for negligible change in substrate concentration for measurement of initial rates, since oxygen can be supplied along the reactor as it is consumed. Based on this concept, a system suitable for kinetic characterization of oxygen dependent enzymes was developed by combining the TiTR with precise liquid and gas supply sys- tems and connecting the outlet of the inner tube to a UV/Vis detector. By means of a switch valve, samples were carried from the injection loop into the detector, where the solution was subjected to flow injection analysis. To demonstrate the performance of the instrument, the well-known enzyme, glucose oxidase (GOx, E.C. 1.1.3.4), was se- lected. The GOx enzyme catalyzes the oxidation of glucose to glucono-d-lactone, using molecular oxygen (which is itself re- duced to hydrogen peroxide). Following the enzymatic reac- tion, glucono-d-lactone is spontaneously hydrolyzed to glucon- ic acid, which formation can be followed spectrophotometri- cally (see Supporting Information). The hydrogen peroxide formed is removed instantaneously by the addition of catalase, which enables its conversion into water and half the stoichio- metric amount of oxygen. The removal of hydrogen peroxide forces the reaction to proceed in a unidirectional manner and also protects GOx from oxidation. GOx has been shown to follow a ping-pong bi-bi reaction mechanism (Scheme 1)[24] for which a rate expression can be derived (Equation (1)). Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a Conse- quently, the reactor can be described by plug-flow behavior, which was used in a method recently reported by Moore and Jensen.[21] In this method, at low residence time, steady-state is obtained and the flow rate is subsequently ramped down. By following the conversion during the ramp, initial rate measure- ments (i.e. concentration-time profiles) are possible without the need to obtain multiple steady-states. Nevertheless, the re- ported Moore and Jensen method requires modification for biocatalysis. Low dispersed flow is very dependent on the dif- fusivity of the solutes, and the large size of enzyme catalysts translates into a two order-of-magnitude lower diffusivity com- pared to small molecules (10@11 cf. 10@9 m2s@1).[22,23] The axial dispersion of enzymes will therefore be much more pro- nounced, indicating that enzymes are more dispersed along the length of the channel compared to the small molecule re- actants and the resulting products. It was therefore necessary to make sure that the enzyme concentration in the entire reac- tor volume remained constant. This was ensured by achieving steady-state with respect to the enzyme concentration and thereafter keeping the enzyme feed concentration constant, in- dependent of the liquid flow rate. In this way, it was assumed that the degree of dispersion would be dependent on the dif- fusion coefficients of the substrate(s) and product(s) alone. The integrated combination of each of the aforementioned devel- opments has led to the establishment of the current instru- ment, which now gives a novel and automated way of kineti- cally characterizing oxygen-dependent enzymes, see Figure 1. The specific details of the setup are described in the Support- ing Information (SI). Recently, the Teflon AF-2400 fluoropolymer,[14] which is char- acterized by high gas permeability, has been used as a mem- brane in the latest development of the so-called Tube-in-Tube Reactor (TiTR) design[15] which has previously proven to be useful for the supply of gaseous substrates to liquid reaction media while retaining the chemical resistance of traditional flu- oropolymers.[16,17] The TiTR is made of an inner Teflon AF-2400 tube encased within an outer PTFE tube with low oxygen per- meability. A mixture of oxygen and nitrogen is supplied in the space between the two tubes, whereby the oxygen can be transferred to the liquid reaction mixture in the inner tube through the membrane. ChemCatChem 2017, 9, 3285 – 3288 Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a 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. 3285 ChemCatChem 2017, 9, 3285 – 3288 Communications the partial pressure), whereas the interfacial effect can only be alleviated by introducing a physical barrier between the gas and the liquid. “tongue” profile of laminar flow, and solute concentrations will thereby only change along the length of the reactor. Conse- quently, the reactor can be described by plug-flow behavior, which was used in a method recently reported by Moore and Jensen.[21] In this method, at low residence time, steady-state is obtained and the flow rate is subsequently ramped down. By following the conversion during the ramp, initial rate measure- ments (i.e. concentration-time profiles) are possible without the need to obtain multiple steady-states. Nevertheless, the re- ported Moore and Jensen method requires modification for biocatalysis. Low dispersed flow is very dependent on the dif- fusivity of the solutes, and the large size of enzyme catalysts translates into a two order-of-magnitude lower diffusivity com- pared to small molecules (10@11 cf. 10@9 m2s@1).[22,23] The axial dispersion of enzymes will therefore be much more pro- nounced, indicating that enzymes are more dispersed along the length of the channel compared to the small molecule re- actants and the resulting products. It was therefore necessary to make sure that the enzyme concentration in the entire reac- tor volume remained constant. This was ensured by achieving steady-state with respect to the enzyme concentration and thereafter keeping the enzyme feed concentration constant, in- dependent of the liquid flow rate. In this way, it was assumed that the degree of dispersion would be dependent on the dif- fusion coefficients of the substrate(s) and product(s) alone. The integrated combination of each of the aforementioned devel- opments has led to the establishment of the current instru- ment, which now gives a novel and automated way of kineti- cally characterizing oxygen-dependent enzymes, see Figure 1. The specific details of the setup are described in the Support- ing Information (SI). “tongue” profile of laminar flow, and solute concentrations will thereby only change along the length of the reactor. www.chemcatchem.org T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3286 Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a Although such a reactor is very useful for conducting oxygen-dependent enzyme reactions (under pressure), we real- ized that a further development was still necessary for the meaningful collection of kinetic data. Laboratory flow reactors typically operate in the laminar flow regime with large axial dispersion, which requires steady-state experiments. Such ex- periments often consume more material over a longer time period and with a lower sampling frequency than those per- formed in equivalent batch apparatus.[18] Recently, a review of Taylor’s work regarding mixing and dispersion[19] has led to the application of low dispersed flow in microreactors.[20] This is a unique regime of laminar flow that occurs only at a microflui- dic scale.[20] In this flow regime, the radial mixing from the center of the tube to the edges is governed solely by diffusion. At the microscale, the diffusion lengths are by definition very small and this will in turn give very short radial mixing times. Low dispersed flow will therefore flatten the well-known r E½ A ¼ kcatS O S O þ KMO S þ KMS O ð1Þ ð1Þ The flow manipulation method applied to produce the equivalent batch data from the setup, requires an accurate de- termination of the reactor volume. Hence, initially, residence time distribution experiments were conducted to determine the volume of the reactor (155:1.8 mL, see Supporting Infor- mation). Next, the results of the flow method were compared with steady-state operation, and it was shown that the setup T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3286 Figure 1. Experimental setup of the Tube-in-Tube Reactor. The three syringe pumps on the left deliver a liquid solution to the inner membrane tube, illustrat- ed by the orange line. Two mass flow controllers are used to vary the gas composition in the range 5–100% O2, supplied to the outer tube. The gas is wetted and heated before entering the reactor to avoid the stripping of water from the inner tube. The gas was fed through an outer tube, made of PTFE. A pressure regulator and a manometer were located at both ends of the two tubes to control the pressure, as well as to ensure an equal or higher pressure on the liquid side of the membrane. Figure 1. Experimental setup of the Tube-in-Tube Reactor. Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley[a The three syringe pumps on the left deliver a liquid solution to the inner membrane tube, illustrat- ed by the orange line. Two mass flow controllers are used to vary the gas composition in the range 5–100% O2, supplied to the outer tube. The gas is wetted and heated before entering the reactor to avoid the stripping of water from the inner tube. The gas was fed through an outer tube, made of PTFE. A pressure regulator and a manometer were located at both ends of the two tubes to control the pressure, as well as to ensure an equal or higher pressure on the liquid side of the membrane. Scheme 1. Cleveland representation of the glucose oxidase ping-pong bi bi mechanism. E denotes the oxidized free form of the enzyme whereas F de- notes the reduced form of the free enzyme. Figure 2. Specific initial reaction rate vs. oxygen concentration in Batch (x) and TiTR (o) at a glucose concentration of 400 mm (blue), 200 mm (red), 100 mm (yellow), and 25 mm (purple). Full lines represent the model fit to the TiTR results. The experiments were carried out at pH 7, 258C and atmos- pheric pressure. The batch data was scaled by a factor of 0.79 to correct for time dependent degradation of the enzyme formulation between the ex- periments, see Supporting Information. Scheme 1. Cleveland representation of the glucose oxidase ping-pong bi bi mechanism. E denotes the oxidized free form of the enzyme whereas F de- notes the reduced form of the free enzyme. indeed produces time-series data even with the addition of a slow diffusing (bio)catalyst (see SI). Finally, to validate the enzyme kinetics measured in the TiTR, equivalent experiments to those carried out in batch by Toftgaard Pedersen and co- workers[25] were conducted. In the batch experiments, the setup used an aerated stirred tank reactor with adjustable oxygen/nitrogen feed. The comparison revealed an excellent correlation between the two systems and the combined results of the validation experiments confirmed that the kinetics de- termined using the TiTR setup are reliable (Figure 2). Figure 2. Specific initial reaction rate vs. oxygen concentration in Batch (x) and TiTR (o) at a glucose concentration of 400 mm (blue), 200 mm (red), 100 mm (yellow), and 25 mm (purple). Full lines represent the model fit to the TiTR results. ChemCatChem 2017, 9, 3285 – 3288 Keywords: automated flow reactor · enzyme catalysis · kinetics · oxidation · tube-in-tube [1] W. Kroutil, H. Mang, K. Edegger, K. Faber, Adv. Synth. Catal. 2004, 346, 125–142. [2] F. Hollmann, I. W. C. E. Arends, K. Buehler, A. Schallmey, B. Behler, Green Chem. 2011, 13, 226. [3] T. Punniyamurthy, S. Velusamy, J. Iqbal, Chem. Rev. 2005, 105, 2329– 2363. Figure 3. Data collected in the TiTR at 1 atm. (0.14–1.3 mm O2) and 6 bar (0.9–7.13 mm O2) at a glucose concentration of 400 mm (blue), 200 mm (red), 100 mm (yellow), and 25 mm (purple). Full lines represent the model fit. Experiments were carried out at pH 7 and 258C. [4] A. Fallah-Araghi, J.-C. Baret, M. Ryckelynck, A. D. Griffiths, Lab Chip 2012, 12, 882–891. [5] U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K. Robins, Nature 2012, 485, 185–194. [6] R. Eisenthal, M. J. Danson, D. W. Hough, Trends Biotechnol. 2007, 25, 247–249. and TiTR, the batch setup requires four full days of labor. Fur- thermore, the small dimensions of the system make it possible to collect one initial rate measurement per 1.4 mL of reaction mixture, which is considerably less than the 150 mL required in the alternative sparged batch setup. [7] D. E. Koshland, Bioorg. Chem. 2002, 30, 211–213. [8] R. J. Fox, M. D. Clay, Trends Biotechnol. 2009, 27, 137–140. [9] R. H. Ringborg, J. M. Woodley, React. Chem. Eng. 2016, 1, 10–22. [10] M. Nordkvist, P. M. Nielsen, J. Villadsen, Biotechnol. Bioeng. 2007, 97, 694–707. [11] M. Ghanem, F. Fan, K. Francis, G. Gadda, Biochemistry 2003, 42, 15179– 15188. In summary, we have developed and validated an automat- ed flow reactor system that rapidly and accurately determines the kinetics of oxygen-dependent enzymes. The tool allows perfect control of the oxygen concentration in solution, which by pressurizing the system can enable values that are up to 25-fold higher than the values achievable by using merely air under atmospheric conditions. Operation in the low dispersed flow regime allowed the generation of time-series data with an enzymatic catalyst, despite its low diffusivity, and the resulting data were in good agreement with experiments conducted in a batch system. The system is capable of characterizing the ki- netics of any enzyme within the oxidoreductase class (EC 1), for which reactions frequently result in changes to the UV- spectra, to enable facile quantification of conversion. Keywords: automated flow reactor · enzyme catalysis · kinetics · oxidation · tube-in-tube The ap- plication is however not limited to oxygen-dependent en- zymes alone, but can in principle be used to study many other enzymes using gaseous substrates, such as hydrogenases (using H2),[26] formate dehydrogenases (using CO2)[27] or meth- ane monooxygenases (using CH4).[28] The tool presented here could introduce kinetic characterization of oxidoreductases into the catalyst development cycle, where biocatalytic reac- tion engineering can be used to guide both process and pro- tein engineering.[9,29] The need to improve this development cycle further is particularly important to facilitate the wider and more effective implementation of biocatalytic reactions, especially in the pharmaceutical industry.[30] [12] L. Pollegioni, B. Langkau, W. Tischer, S. Ghisla, M. S. Pilone, J. Biol. Chem. 1993, 268, 13850–13857. 13] A. S. Bommarius, A. Karau, Biotechnol. Prog. 2005, 21, 1663–167 [14] P. R. Resnick, W. H. Buck in Mod. Fluoropolymers, Wiley, Chichester, 1997, pp. 397–419. [15] A. Polyzos, M. O. Brien, T. P. Petersen, I. R. Baxendale, S. V. Ley, Angew. Chem. Int. Ed. 2011, 50, 1190–1193; Angew. Chem. 2011, 123, 1222– 1225. [16] M. O’Brien, N. Taylor, A. Polyzos, I. R. Baxendale, S. V. Ley, Chem. Sci. 2011, 2, 1250. [17] B. Tomaszewski, A. Schmid, K. Buehler, Org. Process Res. Dev. 2014, 18, 1516–1526. [18] F. E. Valera, M. Quaranta, A. Moran, J. Blacker, A. Armstrong, J. T. Cabral, D. G. Blackmond, Angew. Chem. Int. Ed. 2010, 49, 2478–2485; Angew. Chem. 2010, 122, 2530–2537. [19] G. Taylor, Proc. R. Soc. London Ser. A 1953, 219, 186–203. [20] K. D. Nagy, B. Shen, T. F. Jamison, K. F. Jensen, Org. Process Res. Dev. 2012, 16, 976–981. [21] J. S. Moore, K. F. Jensen, Angew. Chem. Int. Ed. 2014, 53, 470–473; Angew. Chem. 2014, 126, 480–483. [22] E. L. Cussler, Diffusion: Mass Transfer in Fluid Systems, Cambridge Univer- sity Press, 2009. [23] M. E. Young, P. A. Carroad, R. L. Bell, Biotechnol. Bioeng. 1980, 22, 947– 955. [24] Q. H. Gibson, B. E. P. Swoboda, V. Massey, J. Biol. Chem. 1964, 239, 3927–3934. [25] A. Toftgaard Pedersen, T. Carvalho, E. Sutherland, G. Rehn, R. Ashe, J. M. Woodley, Biotechnol. Bioeng. 2017, 114, 1222–1230. [26] L. Lauterbach, O. Lenz, K. A. Vincent, FEBS J. 2013, 280, 3058–3068. [27] J. Shi, Y. Jiang, Z. Jiang, X. Wang, X. Wang, S. Zhang, P. Han, C. Yang, Chem. Soc. Rev. 2015, 44, 5981–6000. [28] S. I. Chan, Y. J. Lu, P. Nagababu, S. Maji, M. C. The authors declare no conflict of interest. Keywords: automated flow reactor · enzyme catalysis · kinetics · oxidation · tube-in-tube Keywords: automated flow reactor · enzyme catalysis · kinetics · oxidation · tube-in-tube Hung, M. M. Lee, I. J. Hsu, P. D. Minh, J. C. H. Lai, K. Y. Ng, S. Ramalingam, S. S. F. Yu, M. K. Chan, Angew. Chem. Int. Ed. 2013, 52, 3731–3735; Angew. Chem. 2013, 125, 3819–3823. Communications Conflict of interest Figure 3. Data collected in the TiTR at 1 atm. (0.14–1.3 mm O2) and 6 bar (0.9–7.13 mm O2) at a glucose concentration of 400 mm (blue), 200 mm (red), 100 mm (yellow), and 25 mm (purple). Full lines represent the model fit. Experiments were carried out at pH 7 and 258C. ChemCatChem 2017, 9, 3285 – 3288 T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 288 Rolf H. Ringborg+,[a, b] Asbjørn Toftgaard Pedersen+,[a] and John M. Woodley*[a The experiments were carried out at pH 7, 258C and atmos- pheric pressure. The batch data was scaled by a factor of 0.79 to correct for time dependent degradation of the enzyme formulation between the ex- periments, see Supporting Information. Table 1. Parameter estimations based on different experimental data. Pressure is given as absolute pressure. Parameter Batch reactor TiTR TiTR (1 atm) (1 atm) (1 atm+6 bar) kcat [mmolmin@1 mg@1][a] 17.58:0.62[b] 17.78:1.39 17.82:0.47 KMO [mm] 0.45:0.04 0.51:0.09 0.52:0.03 KMS [mm] 73.1:6.87 75.2:9.38 74.57:5.55 [a] Based on milligrams of liquid formulation [b] The batch data is scaled by a factor 0.79 to correct for time dependent degradation of the enzyme formulation between the experiments, see SI. Table 1. Parameter estimations based on different experimental data. Pressure is given as absolute pressure. The fit of Equation (1) to these data revealed a relatively high Michaelis constant of 0.52 mm for oxygen (Table 1), which is also obtained from the unsaturated enzyme kinetics ob- served at high glucose concentrations and atmospheric pres- sure (Figure 2). It is generally accepted, that to reliably quantify Michaelis constants it is necessary to measure enzyme kinetics in a sufficiently large range of substrate concentrations, com- prising values that are 5-fold (as a minimum, and preferably 10-fold) higher and lower than the true KM. In the TiTR setup, this was achieved by increasing the operating pressure of the setup to 6 bar to increase the maximum dissolved oxygen con- centration to 7.13 mm (using pure O2 at 258C). Enzyme satura- tion was thereby obtained even at the highest concentration of glucose (Figure 3), enabling a more reliable prediction of all the kinetic parameters (Table 1). [a] Based on milligrams of liquid formulation [b] The batch data is scaled by a factor 0.79 to correct for time dependent degradation of the enzyme formulation between the experiments, see SI. The TiTR setup was fully automated and computer con- trolled, thereby enabling characterization of an oxygen-depen- dent enzyme within 24 hours with minimal manual labor. While the preparation of solutions is identical for both batch 3287 85 – 3288 www.chemcatchem.org T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3287 www.chemcatchem.org T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 287 Acknowledgements [29] J. M. Woodley, Curr. Opin. Chem. Biol. 2013, 17, 310–316. The research leading to these results has received funding from the European Union’s Seventh Framework Programme for re- search, technological development and demonstration under grant agreement n8 613849 supporting the project BIOOX (A.T.P.). The authors acknowledge Novozymes A/S (Bagsværd, DK) for kindly supplying the enzyme used for this research. [30] M. D. Truppo, ACS Med. Chem. Lett. 2017, https://doi.org/10.1021/acs- medchemlett.7b00114. Manuscript received: May 16, 2017 Revised manuscript received: June 21, 2017 Accepted manuscript online: June 27, 2017 Version of record online: August 10, 2017 T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3288 ChemCatChem 2017, 9, 3285 – 3288 3288 www.chemcatchem.org
https://openalex.org/W2220858411	https://europepmc.org/articles/pmc3998472?pdf=render	English	null	3-(4-Hexyloxyphenyl)-1,2,4-triazolo[3,4-<i>b</i>]benzothiazole	Acta crystallographica. Section E	2,014	cc-by	3,323	Dieter Schollmeyer and Heiner Detert* 3441 reﬂections with I > 2(I) Rint = 0.074 University Mainz, Duesbergweg 10-14, 55099 Mainz, Germany Correspondence e-mail: detert@uni-mainz.de Reﬁnement R[F 2 > 2(F 2)] = 0.047 wR(F 2) = 0.119 S = 1.04 4225 reﬂections Received 28 January 2014; accepted 29 January 2014 227 parameters H-atom parameters constrained max = 0.30 e A˚ 3 min = 0.23 e A˚ 3 Key indicators: single-crystal X-ray study; T = 193 K; mean (C–C) = 0.002 A˚; R factor = 0.047; wR factor = 0.119; data-to-parameter ratio = 18.6. Data collection: X-AREA (Stoe & Cie, 2011); cell reﬁnement: X- AREA; data reduction: X-RED (Stoe & Cie, 2011); program(s) used to solve structure: SIR97 (Altomare et al., 1999); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: PLATON (Spek, 2009); software used to prepare material for publication: PLATON. The title compound, C20H21N3OS, was prepared by Huisgen reaction of 5-(4-hexyloxyphenyl)tetrazole and chlorobenzo- thiazole. The essentially planar benzothiazolotriazole frame- work [maximum deviation from the mean plane of 0.077 (1) A˚ for the bridgehead N atom] and the phenyl ring form a dihedral angle of 53.34 (5). The hexyloxy chain adopts a gauche–all-anti conformation. The intracentroid separation of 3.7258 (8) A˚ between the triazole and benzene rings is the closest contact between individual molecules in the crystal. Supporting information for this paper is available from the IUCr electronic archives (Reference: NC2323). Related literature Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L., Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115–119. For related benzothiazolotriazoles, see: Butler et al. (1972); Reynolds & van Allan (1959). For triazolo-annulation via tetrazoles, see: Christiano et al. (2008). For the Huisgen reaction, see: Huisgen et al. (1960,1961). For the structures of related triazolo-annulated heterocycles, see: Preis et al. (2011a,b); Herget et al. (2013); Puviarasnan et al. (1999). pp y Butler, R. N., O’Sullivan, P. & Scott, L. F. (1972). J. Chem. Soc. Perkin Trans. 1, pp. 1519–1523. Christiano, R., Gallardo, H., Bortoluzzi, A. J., Bechtold, I. H., Campos, C. E. M. & Longo, L. R. (2008). Chem. Commun. pp. 5134–5136. Herget, K., Schollmeyer, D. & Detert, H. (2013). Acta Cryst. E69, o365–o366. Huisgen, R., Sturm, H. J. & Markgraf, J. H. (1960). Chem. Ber. 93, 2106–2124. Huisgen, R., Sturm, H. J. & Seidel, M. (1961). Chem. Ber. 94, 1555–1562. Preis, J., Schollmeyer, D. & Detert, H. (2011a). Acta Cryst. E67, o987. Preis, J., Schollmeyer, D. & Detert, H. (2011b). Acta Cryst. E67, o2551. Puviarasan, K., Govindasamy, L., Shanmuga Sundara Raj, S., Velmurugan, D., Jayanthi, G. & Fun, H.-K. (1999). Acta Cryst. C55, 948–951. Reynolds, G. A. & van Allan, J. A. (1959). J. Org. Chem. 24, 1478–1486. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Stoe & Cie (2011). X-AREA and X-RED. Stoe & Cie, Darmstadt, Germany. Christiano, R., Gallardo, H., Bortoluzzi, A. J., Bechtold, I. H., Campos, C. E. M. & Longo, L. R. (2008). Chem. Commun. pp. 5134–5136. Herget, K., Schollmeyer, D. & Detert, H. (2013). Acta Cryst. E69, o365–o366. Huisgen, R., Sturm, H. J. & Markgraf, J. H. (1960). Chem. Ber. 93, 2106–2124. Huisgen, R., Sturm, H. J. & Seidel, M. (1961). Chem. Ber. 94, 1555–1562. Preis, J., Schollmeyer, D. & Detert, H. (2011a). Acta Cryst. E67, o987. Preis, J., Schollmeyer, D. & Detert, H. (2011b). Acta Cryst. E67, o2551. Puviarasan, K., Govindasamy, L., Shanmuga Sundara Raj, S., Velmurugan, D., uviarasan, K., Govindasamy, L., Shanmuga Sundara Raj, S., Ve Puviarasan, K., Govindasamy, L., Shanmuga Sundara Raj, S., Velmurugan, D., Jayanthi, G. & Fun, H.-K. (1999). Acta Cryst. C55, 948–951 Jayanthi, G. & Fun, H.-K. (1999). Acta Cryst. C55, 948–951. Reynolds, G. A. & van Allan, J. A. (1959). J. Org. Chem. 24, 1478–1486. 3-(4-Hexyloxyphenyl)-1,2,4-triazolo- [3,4-b]benzothiazole Dieter Schollmeyer and Heiner Detert* University Mainz, Duesbergweg 10-14, 55099 Mainz, Germany Correspondence e-mail: detert@uni-mainz.de Received 28 January 2014; accepted 29 January 2014 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Experimental Crystal data C20H21N3OS Mr = 351.46 Orthorhombic, Pbcn a = 10.7369 (4) A˚ b = 9.1770 (3) A˚ c = 35.6567 (11) A˚ V = 3513.3 (2) A˚ 3 Z = 8 Mo K radiation = 0.20 mm1 T = 193 K 0.50 0.20 0.20 mm 3-(4-Hexyloxyphenyl)-1,2,4-triazolo- [3,4-b]benzothiazole Data collection Stoe IPDS 2T diffractometer 26869 measured reﬂections 4225 independent reﬂections Acta Cryst. (2014). E70, o247 organic compounds 3. Refinement Hydrogen atoms attached to carbons were placed at calculated positions with C—H = 0.95 Å (aromatic) or 0.98–0.99 Å (sp3 C-atom). All H atoms were refined in the riding-model approximation with isotropic displacement parameters (set at 1.2–1.5 times of the Ueq of the parent atom). 2. Experimental The title compound was prepared by adding chlorobenzothiazole (0.49 g, 2.75 mmol) to a stirred solution of 5-(4-hexyl- oxyphenyl)tetrazole (0.67 g, 2.75 mmol) and collidine (0.5 ml) in xylenes (12 ml). The mixture was stirred for 15 h at ambient temperature and heated to 392 K for 24 h. The cooled solution was filtered, washed with water (30 ml), dried (CaCl2) and concentrated. Chromatography on silica gel using toluene / ethyl acetate 3 / 7 (Rf = 0.15) as an eluent yielded 0.66 g of the pure title compound (68%). Recrystallization from methanol / dichloromethane gave off-white crystals with m.p. = 400 K 1. Comment The Huisgen reaction of tetrazoles and 2-chloroazines is a versatile method for the synthesis of 1,2,4-triazolo-annulated azines see: Preis et al. (2011a, 2011b), Herget et al. (2013), and Christiano et al. (2008). This method is also applicable to azoles with active chlorine and this structure confirms the corresponding [3,4-b]- annulation. The essentially planar benzothiazolotriazole framework of title compound C20H21N3OS (max. deviation 0.077 Å from mean plane at N(8) and the planar phenyl ring open a dihedral angle of 53.34 (5)°. The structural features of the π-system are very similar to a derivative lacking the hexyloxy group, see Puviarasnan et al. (1999). The O19—C20 bond is nearly coplanar with the mean plane of the phenyl ring (torsion angle: 6.6°) and the hexyl chain shows a gauche-all-anti konformation. A minimal distance of 3.73 Å between neighbouring molecules was found for the centroids of the triazole and the benzo-ring. Related literature Sheldrick G M (2008) A t C t A64 112 122 y , , ( ) y , Reynolds, G. A. & van Allan, J. A. (1959). J. Org. Chem. 24, 1478–1486. Reynolds, G. A. & van Allan, J. A. (1959). J. Org. Chem. 24, y ( ) Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. ( ) y Spek, A. L. (2009). Acta Cryst. D65, 148–155. Stoe & Cie (2011). X-AREA and X-RED. Stoe & Cie, Darmstadt, Germany. o247 Acta Cryst. (2014). E70, o247 Schollmeyer and Detert o247 doi:10.1107/S1600536814002153 Schollmeyer and Detert o supplementary materials Acta Cryst. (2014). E70, o247 [doi:10.1107/S1600536814002153] Acta Cryst. (2014). E70, o247 [doi:10.1107/S1600536814002153] Acta Cryst. (2014). E70, o247 [doi:10.1107/S1600536814002153] Acta Cryst. (2014). E70, o247 [doi:10.1107/S1600536814002153] Figure 1 Figure 1 Crystal structure of the title compound with labeling and displacement ellipsoids drawn at the 50% probability level. 3-(4-Hexyloxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole Computing details Data collection: X-AREA (Stoe & Cie, 2011); cell refinement: X-AREA (Stoe & Cie, 2011); data reduction: X-RED (Stoe & Cie, 2011); program(s) used to solve structure: SIR97 (Altomare et al., 1999); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: PLATON (Spek, 2009); software used to prepare material for publication: PLATON (Spek, 2009). sup-1 Acta Cryst. (2014). E70, o247 supplementary materials Figure 1 Special details Experimental. 1H-NMR (CDCl3): δ = 7.66 (m, 3 H); 7.51 (d, 1 H, J = 7.5 Hz), 7.35 ("t", 1 H), 7.29 ("t", 1H), 7.06 (d, 2 H, J = 8 Hz), 4.05, t, 2 H), 1.83 (qui, 2 H), 1.48 (qui, 2 H), 1.33 (m, 4 H), 0.90 ("t", 3H). 13C-NMR(CDCl3): δ = 156.0, 150.7, 154.3, 127.5, 125.5 (2c), 125.0, 121.2, 121.1, 119.7, 113.5, 109.9, 109.2, 63.2, 26.5, 24.1, 20.6, 17.5, 8.9. FD— MS:351.4 (M+H+). UV-Vis: dichloromethane: λmax = 260 nm (log ε = 4.11), λmax = 298 nm (log ε = 3.85); cyclohexane: λmax = 261 nm, λmax = 298 nm; λmax = 298 nm; ethanol: λmax = 258 nm, λmax = 295 nm; fluorescence: dichloromethane: λmax = 356 nm; cyclohexane: λmax = 333 nm; ethanol: λmax = 358 nm. Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. 3-(4-Hexyloxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole Crystal data C20H21N3OS Mr = 351.46 Orthorhombic, Pbcn Hall symbol: -P 2n 2ab a = 10.7369 (4) Å b = 9.1770 (3) Å c = 35.6567 (11) Å V = 3513.3 (2) Å3 Z = 8 F(000) = 1488 Dx = 1.329 Mg m−3 Melting point: 400 K Mo Kα radiation, λ = 0.71073 Å Cell parameters from 32877 reflections θ = 2.2–33.7° µ = 0.20 mm−1 T = 193 K Needle, colourless 0.50 × 0.20 × 0.20 mm Data collection Stoe IPDS 2T diffractometer Radiation source: sealed X-ray tube, 12 x 0.4 mm long-fine focus Graphite monochromator Detector resolution: 6.67 pixels mm-1 rotation method scans 26869 measured reflections 4225 independent reflections 3441 reflections with I > 2σ(I) Rint = 0.074 θmax = 28.0°, θmin = 2.9° h = −14→14 k = −8→12 l = −47→38 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.047 wR(F2) = 0.119 S = 1.04 4225 reflections 227 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0653P)2 + 0.9425P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.23 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0653P)2 + 0.9425P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.23 e Å−3 sup-2 Acta Cryst. (2014). E70, o247 supplementary materials Acta Cryst. (2014). E70, o247 Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq S1 0.41618 (4) 0.26300 (4) 0.696695 (10) 0.03581 (13) C2 0.40821 (12) 0.43744 (15) 0.67607 (4) 0.0304 (3) C3 0.41068 (14) 0.57003 (18) 0.69481 (4) 0.0364 (3) H3 0.4124 0.5737 0.7214 0.044 C4 0.41053 (13) 0.69677 (17) 0.67376 (5) 0.0374 (3) H4 0.4115 0.7884 0.6861 0.045* C5 0.40897 (13) 0.69200 (16) 0.63482 (5) 0.0340 (3) H5 0.4109 0.7804 0.6210 0.041* C6 0.40465 (12) 0.55988 (15) 0.61587 (4) 0.0299 (3) H6 0.4030 0.5564 0.5892 0.036* C7 0.40282 (11) 0.43353 (14) 0.63696 (4) 0.0260 (3) N8 0.40149 (10) 0.28738 (12) 0.62443 (3) 0.0263 (2) C9 0.40200 (12) 0.20746 (15) 0.59169 (4) 0.0280 (3) N10 0.41399 (11) 0.06852 (13) 0.60013 (4) 0.0348 (3) N11 0.42111 (12) 0.05377 (13) 0.63912 (4) 0.0361 (3) C12 0.41293 (12) 0.18574 (15) 0.65223 (4) 0.0304 (3) C13 0.38612 (12) 0.26957 (15) 0.55412 (4) 0.0280 (3) C14 0.46740 (13) 0.23437 (15) 0.52481 (4) 0.0311 (3) H14 0.5331 0.1670 0.5291 0.037* C15 0.45328 (13) 0.29627 (16) 0.48985 (4) 0.0323 (3) H15 0.5089 0.2710 0.4702 0.039* C16 0.35734 (12) 0.39615 (15) 0.48326 (4) 0.0289 (3) C17 0.27419 (12) 0.42904 (15) 0.51209 (4) 0.0307 (3) H17 0.2073 0.4947 0.5077 0.037* C18 0.28918 (12) 0.36596 (15) 0.54696 (4) 0.0304 (3) H18 0.2321 0.3889 0.5664 0.036* O19 0.35160 (9) 0.45394 (11) 0.44817 (3) 0.0345 (2) C20 0.25760 (14) 0.56334 (16) 0.44224 (4) 0.0354 (3) H20A 0.2658 0.6407 0.4614 0.043* H20B 0.1739 0.5191 0.4448 0.043* C21 0.27167 (14) 0.62833 (16) 0.40378 (4) 0.0367 (3) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2014). E70, o247 supplementary materials supplementary materials pp y 1.396 (2) C17—H17 0.9500 1.384 (2) C18—H18 0.9500 0.9500 O19—C20 1.4392 (17) 1.389 (2) C20—C21 1.503 (2) 0.9500 C20—H20A 0.9900 1.389 (2) C20—H20B 0.9900 0.9500 C21—C22 1.521 (2) 1.382 (2) C21—H21A 0.9900 0.9500 C21—H21B 0.9900 1.4137 (17) C22—C23 1.517 (2) 1.3667 (18) C22—H22A 0.9900 1.3787 (18) C22—H22B 0.9900 1.3164 (18) C23—C24 1.515 (2) 1.466 (2) C23—H23A 0.9900 1.399 (2) C23—H23B 0.9900 1.3012 (19) C24—C25 1.512 (2) 1.3897 (19) C24—H24A 0.9900 1.399 (2) C24—H24B 0.9900 1.378 (2) C25—H25A 0.9800 0.9500 C25—H25B 0.9800 1.399 (2) C25—H25C 0.9800 0.9500 89.37 (7) C18—C17—H17 120.1 120.26 (13) C16—C17—H17 120.1 126.43 (12) C17—C18—C13 121.31 (13) 113.28 (11) C17—C18—H18 119.3 118.38 (14) C13—C18—H18 119.3 120.8 C16—O19—C20 116.03 (11) 120.8 O19—C20—C21 109.90 (12) 121.03 (14) O19—C20—H20A 109.7 119.5 C21—C20—H20A 109.7 119.5 O19—C20—H20B 109.7 120.94 (14) C21—C20—H20B 109.7 119.5 H20A—C20—H20B 108.2 119.5 C20—C21—C22 115.56 (12) 117.91 (14) C20—C21—H21A 108.4 121.0 C22—C21—H21A 108.4 121.0 C20—C21—H21B 108.4 121.42 (13) C22—C21—H21B 108.4 128.61 (13) H21A—C21—H21B 107.5 109.90 (12) C23—C22—C21 111.77 (13) 104.52 (12) C23—C22—H22A 109.3 114.67 (12) C21—C22—H22A 109.3 140.56 (12) C23—C22—H22B 109.3 108.80 (13) C21—C22—H22B 109.3 126.64 (13) H22A—C22—H22B 107.9 124.51 (12) C24—C23—C22 114.37 (13) 109.03 (12) C24—C23—H23A 108.7 C2—C7 1.396 (2) C17—H17 0.9500 C3—C4 1.384 (2) C18—H18 0.9500 C3—H3 0.9500 O19—C20 1.4392 (17) C4—C5 1.389 (2) C20—C21 1.503 (2) C4—H4 0.9500 C20—H20A 0.9900 C5—C6 1.389 (2) C20—H20B 0.9900 C5—H5 0.9500 C21—C22 1.521 (2) C6—C7 1.382 (2) C21—H21A 0.9900 C6—H6 0.9500 C21—H21B 0.9900 C7—N8 1.4137 (17) C22—C23 1.517 (2) N8—C12 1.3667 (18) C22—H22A 0.9900 N8—C9 1.3787 (18) C22—H22B 0.9900 C9—N10 1.3164 (18) C23—C24 1.515 (2) C9—C13 1.466 (2) C23—H23A 0.9900 N10—N11 1.399 (2) C23—H23B 0.9900 N11—C12 1.3012 (19) C24—C25 1.512 (2) C13—C18 1.3897 (19) C24—H24A 0.9900 C13—C14 1.399 (2) C24—H24B 0.9900 C14—C15 1.378 (2) C25—H25A 0.9800 C14—H14 0.9500 C25—H25B 0.9800 C15—C16 1.399 (2) C25—H25C 0.9800 C15—H15 0.9500 C12—S1—C2 89.37 (7) C18—C17—H17 120.1 C3—C2—C7 120.26 (13) C16—C17—H17 120.1 C3—C2—S1 126.43 (12) C17—C18—C13 121.31 (13) C7—C2—S1 113.28 (11) C17—C18—H18 119.3 C4—C3—C2 118.38 (14) C13—C18—H18 119.3 C4—C3—H3 120.8 C16—O19—C20 116.03 (11) C2—C3—H3 120.8 O19—C20—C21 109.90 (12) C3—C4—C5 121.03 (14) O19—C20—H20A 109.7 C3—C4—H4 119.5 C21—C20—H20A 109.7 C5—C4—H4 119.5 O19—C20—H20B 109.7 C6—C5—C4 120.94 (14) C21—C20—H20B 109.7 C6—C5—H5 119.5 H20A—C20—H20B 108.2 C4—C5—H5 119.5 C20—C21—C22 115.56 (12) C7—C6—C5 117.91 (14) C20—C21—H21A 108.4 C7—C6—H6 121.0 C22—C21—H21A 108.4 C5—C6—H6 121.0 C20—C21—H21B 108.4 C6—C7—C2 121.42 (13) C22—C21—H21B 108.4 C6—C7—N8 128.61 (13) H21A—C21—H21B 107.5 C2—C7—N8 109.90 (12) C23—C22—C21 111.77 (13) C12—N8—C9 104.52 (12) C23—C22—H22A 109.3 C12—N8—C7 114.67 (12) C21—C22—H22A 109.3 C9—N8—C7 140.56 (12) C23—C22—H22B 109.3 N10—C9—N8 108.80 (13) C21—C22—H22B 109.3 N10—C9—C13 126.64 (13) H22A—C22—H22B 107.9 N8—C9—C13 124.51 (12) C24—C23—C22 114.37 (13) C9—N10—N11 109.03 (12) C24—C23—H23A 108.7 sup-5 Acta Cryst. supplementary materials H21A 0.3565 0.6698 0.4016 0.044 H21B 0.2118 0.7098 0.4013 0.044* C22 0.25134 (13) 0.52382 (17) 0.37123 (4) 0.0349 (3) H22A 0.3231 0.4564 0.3695 0.042* H22B 0.1757 0.4650 0.3760 0.042* C23 0.23673 (14) 0.60407 (17) 0.33430 (4) 0.0368 (3) H23A 0.1652 0.6716 0.3364 0.044* H23B 0.3123 0.6635 0.3299 0.044* C24 0.21655 (17) 0.50588 (19) 0.30070 (5) 0.0451 (4) H24A 0.2927 0.4472 0.2965 0.054* H24B 0.1475 0.4377 0.3063 0.054* C25 0.1863 (2) 0.5888 (2) 0.26520 (5) 0.0612 (5) H25A 0.1085 0.6430 0.2687 0.092* H25B 0.1768 0.5200 0.2444 0.092* H25C 0.2540 0.6570 0.2596 0.092* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 S1 0.0409 (2) 0.0353 (2) 0.0313 (2) 0.00007 (15) −0.00034 (14) 0.00440 (14) C2 0.0281 (6) 0.0311 (7) 0.0319 (7) 0.0002 (5) −0.0004 (5) −0.0004 (6) C3 0.0350 (7) 0.0393 (8) 0.0350 (8) −0.0002 (6) −0.0015 (6) −0.0103 (6) C4 0.0349 (7) 0.0303 (7) 0.0471 (9) 0.0004 (6) −0.0036 (6) −0.0129 (7) C5 0.0333 (7) 0.0239 (7) 0.0449 (8) −0.0005 (5) −0.0033 (6) −0.0039 (6) C6 0.0291 (6) 0.0258 (7) 0.0348 (7) −0.0019 (5) −0.0016 (5) −0.0013 (6) C7 0.0228 (6) 0.0231 (6) 0.0322 (7) −0.0009 (5) 0.0001 (5) −0.0043 (5) N8 0.0271 (5) 0.0215 (5) 0.0302 (6) −0.0011 (4) 0.0003 (4) −0.0009 (4) C9 0.0250 (6) 0.0237 (6) 0.0353 (7) −0.0015 (5) −0.0006 (5) −0.0060 (5) N10 0.0345 (6) 0.0243 (6) 0.0455 (7) −0.0005 (5) −0.0050 (5) −0.0035 (5) N11 0.0385 (6) 0.0255 (6) 0.0443 (7) −0.0007 (5) −0.0040 (5) 0.0030 (5) C12 0.0283 (6) 0.0267 (7) 0.0360 (7) −0.0010 (5) −0.0005 (5) 0.0041 (6) C13 0.0275 (6) 0.0249 (6) 0.0315 (7) −0.0025 (5) −0.0028 (5) −0.0063 (5) C14 0.0270 (6) 0.0281 (7) 0.0382 (7) 0.0043 (5) −0.0008 (6) −0.0067 (6) C15 0.0304 (7) 0.0306 (7) 0.0358 (7) 0.0030 (5) 0.0038 (6) −0.0060 (6) C16 0.0286 (6) 0.0257 (6) 0.0324 (7) −0.0016 (5) −0.0025 (5) −0.0061 (5) C17 0.0267 (6) 0.0292 (7) 0.0361 (7) 0.0037 (5) −0.0033 (5) −0.0085 (6) C18 0.0264 (6) 0.0314 (7) 0.0334 (7) 0.0008 (5) 0.0008 (5) −0.0077 (6) O19 0.0360 (5) 0.0331 (5) 0.0343 (5) 0.0066 (4) 0.0009 (4) −0.0016 (4) C20 0.0336 (7) 0.0292 (7) 0.0435 (8) 0.0034 (5) 0.0009 (6) 0.0006 (6) C21 0.0346 (7) 0.0274 (7) 0.0482 (8) −0.0013 (6) 0.0007 (6) 0.0057 (6) C22 0.0319 (7) 0.0290 (7) 0.0437 (8) −0.0024 (5) −0.0040 (6) 0.0081 (6) C23 0.0369 (7) 0.0303 (7) 0.0431 (8) 0.0024 (6) 0.0039 (6) 0.0081 (6) C24 0.0515 (9) 0.0386 (9) 0.0452 (9) −0.0031 (7) 0.0014 (7) 0.0053 (7) C25 0.0812 (14) 0.0626 (12) 0.0398 (9) 0.0116 (11) 0.0092 (9) 0.0047 (9) Geometric parameters (Å º) Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary materials (2014). E70, o247 supplementary materials supplementary materials pp y C12—N11—N10 105.28 (12) C22—C23—H23A 108.7 N11—C12—N8 112.37 (13) C24—C23—H23B 108.7 N11—C12—S1 134.96 (12) C22—C23—H23B 108.7 N8—C12—S1 112.65 (10) H23A—C23—H23B 107.6 C18—C13—C14 118.47 (13) C25—C24—C23 113.19 (15) C18—C13—C9 120.17 (12) C25—C24—H24A 108.9 C14—C13—C9 121.35 (12) C23—C24—H24A 108.9 C15—C14—C13 120.78 (13) C25—C24—H24B 108.9 C15—C14—H14 119.6 C23—C24—H24B 108.9 C13—C14—H14 119.6 H24A—C24—H24B 107.8 C14—C15—C16 120.21 (13) C24—C25—H25A 109.5 C14—C15—H15 119.9 C24—C25—H25B 109.5 C16—C15—H15 119.9 H25A—C25—H25B 109.5 O19—C16—C17 124.37 (12) C24—C25—H25C 109.5 O19—C16—C15 116.32 (12) H25A—C25—H25C 109.5 C17—C16—C15 119.31 (13) H25B—C25—H25C 109.5 C18—C17—C16 119.89 (12) C12—S1—C2—C3 −177.71 (13) C7—N8—C12—N11 −174.91 (11) C12—S1—C2—C7 0.27 (10) C9—N8—C12—S1 179.33 (9) C7—C2—C3—C4 −1.4 (2) C7—N8—C12—S1 3.92 (14) S1—C2—C3—C4 176.40 (11) C2—S1—C12—N11 176.13 (15) C2—C3—C4—C5 −0.5 (2) C2—S1—C12—N8 −2.34 (10) C3—C4—C5—C6 1.5 (2) N10—C9—C13—C18 128.50 (15) C4—C5—C6—C7 −0.4 (2) N8—C9—C13—C18 −48.64 (19) C5—C6—C7—C2 −1.54 (19) N10—C9—C13—C14 −51.7 (2) C5—C6—C7—N8 −178.36 (12) N8—C9—C13—C14 131.13 (14) C3—C2—C7—C6 2.5 (2) C18—C13—C14—C15 1.4 (2) S1—C2—C7—C6 −175.59 (10) C9—C13—C14—C15 −178.40 (13) C3—C2—C7—N8 179.88 (12) C13—C14—C15—C16 0.3 (2) S1—C2—C7—N8 1.76 (13) C14—C15—C16—O19 178.83 (13) C6—C7—N8—C12 173.48 (13) C14—C15—C16—C17 −1.9 (2) C2—C7—N8—C12 −3.63 (15) O19—C16—C17—C18 −179.06 (12) C6—C7—N8—C9 0.5 (2) C15—C16—C17—C18 1.7 (2) C2—C7—N8—C9 −176.62 (14) C16—C17—C18—C13 0.0 (2) C12—N8—C9—N10 −0.47 (14) C14—C13—C18—C17 −1.6 (2) C7—N8—C9—N10 172.96 (14) C9—C13—C18—C17 178.22 (12) C12—N8—C9—C13 177.11 (12) C17—C16—O19—C20 4.47 (19) C7—N8—C9—C13 −9.5 (2) C15—C16—O19—C20 −176.25 (12) N8—C9—N10—N11 0.29 (15) C16—O19—C20—C21 173.79 (12) C13—C9—N10—N11 −177.22 (12) O19—C20—C21—C22 63.84 (16) C9—N10—N11—C12 0.02 (15) C20—C21—C22—C23 166.39 (12) N10—N11—C12—N8 −0.33 (15) C21—C22—C23—C24 179.82 (13) N10—N11—C12—S1 −178.80 (12) C22—C23—C24—C25 172.79 (15) C9—N8—C12—N11 0.50 (15) Acta Cryst. (2014). E70, o247 sup-6
https://openalex.org/W3196505597	https://www.researchsquare.com/article/rs-853297/latest.pdf	English	null	Early aggressive nutrition at 22–23 weeks gestational age improves weight gain and does not worsen neurological prognosis	Research Square (Research Square)	2,021	cc-by	4,682	Early aggressive nutrition at 22–23 weeks gestational age improves weight gain and does not worsen neurological prognosis University of the ryukyudai kouichi Nakanishi university of the ryukyus What Is Known: There is limited evidence on the effect of early aggressive nutrition in ELBW infants at GA22–23 weeks. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Page 1/14 What Is New: The GA22–23-week EAN group also had better weight gain and EAN did not worsen neurodevelopmental outcomes. Abstract The effect of early aggressive nutrition (EAN) on extremely low birth weight (ELBW) infants is unknown. The purpose of this study was to investigate the effect of EAN on ELBW infants, especially premature neonates of 22–23 weeks gestational age (GA22–23-week). Twenty-eight preterm infants of less than 26 weeks were divided into two groups (GA22–23-week group, 10 infants; GA24–25-week group, 18 infants) and compared. Each preterm infant received more than 3.0 g/kg/day of amino acids in the first day after birth and 1.0 g/kg/day of lipid emulsion from the next day. The GA22–23-week group had significantly smaller head circumference (20.4 ± 1.0 cm vs. 22.2 ± 1.4 cm, P = 0.002) and body weight at birth (539 ± 68 g vs. 697 ± 155 g, P = 0.003), but there were no differences in early postnatal weight loss (10.4% ± 6.3% vs. 8.1% ± 6.3%, P = 0.37), and body weight at 37 weeks postmenstrual age (1906 ± 321 g vs. 2081 ± 379 g, P = 0.17). Blood urea nitrogen levels were higher in the GA22–23-week group (59.7 ± 16.6 mg/dl vs. 45.0 ± 10.8 mg/dl, P = 0.004), but there were no differences in direct-bilirubin, bile acids, and ammonia levels. After discharge, there was no significant difference in developmental quotient at 2 years of age (71.3 ± 15.1 vs. 78.1 ± 22.6, P = 0.20) between the two groups. Conclusion: We suggest that EAN reduces the rate of early postnatal weight loss in ELBW infants and contributes to weight gain until full term age. Introduction Recently, early aggressive nutrition (EAN) for neonatal care has become standard practice. Preterm infants are suddenly cut off from nutritive supply after birth, mainly protein and glucose from the placenta, causing starvation, endogenous protein loss, and development of hypercatabolism [1]. Protein is used for energy with a constant glucose supply in preterm infants because they do not have enough fat and glycogen. Without exogenous protein, 1–2% of endogenous protein per day is lost due to proteolysis. The purpose of EAN is to prevent protein catabolism by administering amino acids (AAs) after birth, to make the subsequent development as close as possible to fetal development, and to improve developmental prognosis [2, 3]. It has been reported that EAN intake in very low birth weight (VLBW) infants can reduce the rate of weight loss and time to regain birthweight, and provide good subsequent Page 2/14 Page 2/14 weight gain [4–6]. It has also been reported that early high protein intake in extremely low birth weight (ELBW) infants improved weight and height growth outcomes [7]. However, there are few reports of EAN targeting ELBW infants between gestational age 22 and 25 (GA22–25) weeks, and the effect is unclear. Furthermore, there are potential complications with EAN, such as long-term growth and neurodevelopment, azotemia, hyperammonemia, and hyper direct bilirubinemia, but studies of these problems are rare in ELBW infants. The purpose of our study was to examine the effects of EAN, including short-term prognosis and complications, and to determine whether beneficial effects could be obtained even for ELBW GA22–25-week infants. Subjects The study design was a retrospective cohort study. All infants admitted to our neonatal intensive care unit (NICU) from January 2015 to December 2019 were included in the analysis. These infants had GA < 26 weeks at birth and reached the postmenstrual age (PMA) of ≥ 37 weeks. Study subjects consisted of 30 preterm infants, and 28 infants were examined. The other two infants died shortly after birth. One of the 28 infants was small for GA (SGA) with a birth weight and height of less than the 10% tile, and was excluded from the analysis of body weight change during hospitalization. The NICU databases were used as information sources: GA, body size (weight, height, head circumference) at birth, presence of SGA, age when the feeding targets (enteral feeds ≥ 100 ml/kg per day, ≥ 160 ml/kg per day) were achieved, body measurements (weight, height, head circumference) at 37 weeks PMA, the ratio of weight less than the 10% tile at 37 weeks PMA, early postnatal weight loss rate, and days to regain birthweight. We also assessed weight change from birth at 41 weeks PMA, short-term prognosis, complications of total parenteral nutrition (TPN), head circumference at 12 and 18 months, developmental quotient (DQ) at 2 years of age, and head circumference and weight at 3 years age. The 2-year-old DQ was assessed using the Enjoji’s development scale. For complications associated with TPN, maximum measurements of blood urea nitrogen (BUN), direct bilirubin (d-bil) and bile acid were recorded from the results of regular blood sampling during hospitalization. Ammonia levels were recorded at 1 week after birth. The endpoint of short-term prognosis was necrotizing enterocolitis (NEC), patent ductus arteriosus (PDA) treated with indomethacin and/or surgery, retinopathy of prematurity (ROP) treated with laser phototherapy, and intraventricular hemorrhage (IVH) grades III–IV. Necrotizing enterocolitis was defined using Bell’s classification stage 2 or more. The PDA was diagnosed clinically or with echocardiography and was considered clinically significant when it required medical treatment. The IVH was defined using Papile’s classification with ultrasonography. The ROP was treated with laser treatment if it had reached stage III of the international classification as diagnosed by an ophthalmologist. We also investigated the introduction rate of home oxygen therapy, home high-flow nasal canula therapy, home continuous positive airway pressure, tracheostomy, and home tube feeding at discharge. Subjects Page 3/14 Page 3/14 Page 3/14 The protocol for EAN at our facility was as follows: >3.0 g/kg/day amino acid (AA) solution along with a glucose infusion rate of 5.5 mg/kg/min using a 10% glucose solution was started in the first days after birth. At day 2 after birth, lipid emulsion was introduced at 1.0 g/kg/day and was increased every day to a maximum of 3.0 g/kg/day. Fifty mL TPN preparations contained 30 mL of AA (provided as Pleamin-P Injection, Fuso Pharmaceutical Industries, Osaka, Japan), 10 mL of 50% glucose solution, 1 ml of 10% sodium chloride solution, 1 mL dipotassium phosphate solution, and 8 mL distilled water. Additionally, a vitamin preparation (provided as MULTAMIN®ฎ FOR INJECTION, AY PHARMACEUTICALS, Tokyo, Japan), and a trace element preparation (provided as Elemenmic Injection, AY PHARMACEUTICALS. Tokyo, Japan) were added. The lipid emulsion (provided as Intralipos Injection 10%, Otsuka Pharmaceutical Factory, Tokyo, Japan) was administered in parallel from the side tube of the central catheter. Fluids were started at a rate of 80–100 ml/kg/day and increased to 150–160 ml/kg/day. For enteral nutrition, breast milk was used at first, and artificial milk was avoided as much as possible until day 5 after birth. The dose of TPN preparations were increased while replacing enteral nutrition with intravenous nutrition, and used in combination until full feeding was reached. Data analysis was divided into two groups, GA22–23 weeks and GA24–25 weeks. The Z score was calculated using the standard physique at birth by gestational age of Japanese. Additionally, body weight was measured daily until 41 weeks PMA, the Z score was calculated, the average value of each body weight was calculated, and the transition of body weight change was shown as data. The data of SGA was excluded. Head circumference and height could not be calculated because weekly measurements were not taken. The data analysis was performed using the JMP software program, version 14 (SAS Institute, Cary, NC, USA). Data for two groups were compared using the Mann-Whitney U test and chi-square test. The level of significance was set at P < 0.05. Results 11.8 ± 4.8 days, P = 0.51) between the two groups. Table 3 shows the short-term prognosis. The prevalence of IVH III–IV (GA22–23 vs. GA24-25; 3 cases vs. 0 case, P = 0.014) and ROP treated with laser phototherapy (8 cases vs. 7 cases, P = 0.037) in the GA22– 23-week group was higher than the GA24–25-week group, but the prevalence of NEC, PDA treated with indomethacin treatment, PDA treated with surgery, home oxygen therapy, home high-flow nasal canula therapy, tracheostomy, and home tube feeding was similar in the two groups. Table 4 shows the complications associated with TPN. The BUN levels were higher (GA22–23 vs. GA24– 25; 59.7 ± 16.6 mg/dl vs. 45.0 ± 10.8 mg/dl, P = 0.004) in the GA22–23-week group, but no differences were observed in levels of d-bil (1.5 ± 0.7 mg/dl vs. 1.6 ± 0.8 mg/dl, P = 0.89), bile acids (40.1 ± 20.2 mg/dl vs. 53.2 ± 23.0 mg/dl, P = 0.11), and ammonia (71.5 ± 7.4 µg/dl vs. 55.8 ± 18.3 µg/dl, P = 0.25). Moreover, there were no cases requiring treatment because of complications. Table 5 shows the progress of the infants after discharge from the NICU. There were no significant differences between the two groups in all endpoints examined, such as 1-year-old head circumference (GA22–23 vs. GA24–25; 43.0 ± 1.2 cm vs. 43.7 ± 1.4 cm, P = 0.13), 1.5-year-old head circumference (44.5 ± 0.9 cm vs. 45.5 ± 1.7 cm, P = 0.11), 2-year-old DQ (71.3 ± 15.1 vs. 78.1 ± 22.6, P = 0.20), 3-year-old head circumference (48.9 ± 0.4 cm vs. 48.2 ± 1.0 cm, P = 0.12), 3-year-old weight (11.4 ± 0.7 kg vs. 12.6 ± 2.4 kg, P = 0.26) and 3-year-old weight Z score (− 1.4 ± 0.5 vs. −0.4 ± 1.6, P = 0.18). No difference in body weight was observed between the two groups at 37 weeks PMA. Figure 1 shows the changes in Z scores during the course of hospitalization and the changes in body weight catch-up. The GA22–23-week group at showed the lowest Z score at 29 to 35 weeks PMA, but then the Z score gradually increased to − 1.0 ± 1.2 SD at 41 weeks PMA. Results Page 4/14 Thirty infants were admitted to our NICU during the study period, and 28 infants were included in the analysis: 10 in the GA22–23-week group and 18 in the GA24–25-week group. The remaining two infants had severe respiratory failure, did not respond to resuscitation, and died within 24 h after birth. Table 1 shows the clinical characteristics of the two groups. The GA22–23-week group had a significantly lower weight at birth (GA22–23 vs. GA24–25; 539 ± 68 g vs. 697 ± 155 g; P = 0.003), head circumference at birth (20.4 ± 1.0 cm vs. 22.2 ± 1.1 cm; P = 0.002), and height at birth (29.3 ± 2.0 cm vs. 30.9 ± 4.0 cm, P = 0.027). However, there were no significant differences in the Z score of body weight (0.13 ± 0.7 vs. −0.38 ± 1.1, P = 0.35) and number of SGA infants at birth (0 case vs. 1 case, P = 0.45). Table 2 shows the progress of infants during hospitalization. There was no difference in the days to reach enteral feeds of 100 ml/kg (GA22–23 vs. GA24–25; 13.0 ± 5.0 days vs. 12.7 ± 3.7 days, P = 0.94), but the days to reach enteral feeds of 160 ml/kg was significantly delayed in the GA22–23-week group (45.4 ± 22.5 days vs. 23.7 ± 9.2 days, P = 0.008). The head circumferences (28.8 ± 1.2 cm vs. 30.4 ± 2.1 cm, P = 0.015) and associated Z scores (− 2.6 ± 0.8 SD vs. −1.5 ± 1.4 SD, P = 0.016) at 37 weeks PMA were different between the two groups, as they were at birth, but the body weights (1906 ± 321 g vs. 2081 ± 379 g, P = 0.17) and associated Z scores (− 2.3 ± 1.0 vs. −1.7 ± 1.3, P = 0.16) at 37 weeks PMA were not different between the two groups. There Page 4/14 Page 4/14 was only one case of SGA at birth in the GA24–25-week group. However, the number of body weights less than 10% tile at 37 weeks PMA increased in both groups, but the difference was not significant (9 cases vs. 11 cases, P = 0.10). Additionally, there were no differences in the rate of early postnatal weight loss (10.4% ± 6.3% vs. 8.1% ± 6.3%, P = 0.37) and the days of regain to birthweight (13.2 ± 5.9 days vs. Results In the GA24–25-week group, the lowest Z score was at 30 weeks PMA, then at 41 weeks PMA the score recovered to − 0.7 ± 1.2 SD (equivalent to the Z score of body weight at birth). Discussion We found that the body weight of the GA22–23-week group was approaching that of the GA24–25-week group after 37 weeks of PMA, even though sufficient enteral nutrition was delayed in the GA22–23-week group than in the GA24–25-week group. Thureen et al. showed that there are at least three phases of faltering growth commonly seen in ELBW infants, as follows: (1) several weeks immediately after birth when neonates are the most fragile, (2) intermediate time period when infants are commonly and slowly advanced to full enteral nutrition (catch-up period), and (3) the post-discharge phase [8]. Also, preterm infants develop a growth deficit during the first few weeks of life, and this deficit may persist and worsen during hospitalization, causing extrauterine growth restriction (EUGR) [9]. Furthermore, the longer the period before complete enteral feeding was achieved, the greater the risk of EUGR [10–12]. Although Page 5/14 ELBW infants are often discharged with undergrowth, catch-up growth is likely to occur during the follow- up period after discharge [13, 14]. It has been established that EUGR leads to a decrease in head circumference and body weight Z score, which affects neurodevelopment. Although EUGR is an unavoidable challenge in ELBW infants, improving growth during hospitalization in the NICU may improve psychomotor development. Our data showed that early postnatal weight loss was not significantly different between the two groups, with a marked improvement in weight gain from 38 to 41 weeks PMA in the GA22–23-week group, and from 37 to 41 weeks PMA in the GA24–25-week group. Moyses et al. reported in both observational studies and randomized controlled trials that early parenteral nutrition significantly shortened the number of days required to regain birth weight without increasing mortality, chronic lung disease, NEC, or IVH [15]. Radmacher et al. reported that early AA administration shortened the number of days to regain birth weight, increased head circumference and weight at discharge, and reduced the risk of EUGR [16]. In a study comparing high-dose and low-dose intake of AAs, high-dose AA intake was similar to the second and third trimesters of pregnancy. [17]. Here, we propose that the AA dose in the current protocol for ELBW infants at GA22–25 weeks reduced the gradient of weight loss during the weight loss period after birth, then led to weight gain during the subsequent catch- up period. Discussion In the catch-up period, the body weight Z scores fell again at 33 and 34 weeks of PMA in the GA22–23-week and GA24–25-week groups, respectively. But in both groups, body weight Z scores then increased or stabilized at 37 weeks PMA, and caught up at 41 weeks PMA. The cause of the decrease in Z scores after 33–34 weeks PMA may reflect the adaptation to a changing extrauterine environment, such as transfer from the incubator to the cot and the change from premature formula milk to formula milk. In this study, there was a marked catch-up of weight gain after 37 weeks PMA, and there was almost no difference in weights between the GA22–23-week and GA24–25-week groups at 41 weeks PMA. Therefore, EAN had a certain effect on weight gain from postnatal to modified maturity in ELBW and GA22–23-week infants. Regarding short-term prognosis, compared with the 2003–2017 perinatal maternal and child medical center network database analysis report, the incidence of ROP requiring treatment was higher, and that of IVH III–IV was lower, in the GA22–23-week group versus the national data. The possibility of EAN as a cause of the high incidence of ROP cannot be denied, but in our hospital, factors other than EAN, such as prematurity of the preterm infant and systemic management, are considered to be strong factors and require further investigation. Additionally, BUN levels were higher in the GA22–23-week group than those in the GA24–25-week group, and levels in both groups were higher than the normal range, but no treatment was needed in all cases. Balakrishnan et al. showed that there was a positive association between protein loading and BUN levels, and BUN levels improved over time [18]. It was concluded that concerns about metabolic disorders due to early protein administration in ELBW were not justified. However, further research is needed to determine if AA intake is safe and effective. One limitation of this study is that a multivariate analysis was not performed because of the small sample sizes. Regarding the medium- to long-term prognosis, no significant differences in head circumference, body weight, and 2-year-old DQ were observed, but ongoing detailed examinations are still required Page 6/14 Page 6/14 In conclusion, EAN reduced the rate of early postnatal weight loss in very preterm infants, did not cause serious complications, and appeared to contribute to weight gain similar to the fetal period until the time of modified maturity. Discussion In conclusion, EAN reduced the rate of early postnatal weight loss in very preterm infants, did not cause serious complications, and appeared to contribute to weight gain similar to the fetal period until the time of modified maturity. Authors’ contributions MT: data collection, data analysis, manuscript writing. TY: data analysis, data management, manuscript editing, project development. HG: data collection. KN: project development, manuscript editing. Abbreviations AAs, Amino acids; CPAP, Continuous positive airway pressure; DQ, Developmental Quotient; EAN, Early aggressive nutrition; ELBW, Extremely low birth weight, GA; Gestational age; IVH, Intraventricular hemorrhage; NEC, Necrotizing enterocolitis; NICU, Neonatal intensive care unit; PDA, Patent ductus arteriosus; PMA, Postmenstrual age; ROP, Retinopathy of prematurity; SGA, Small for gestational age; TPN, Total parenteral nutrition; VLBW, Very low birth weight Ethics approval The study was conducted with approval of the Medical Research Ethics Review for people at the University of Ryukyus hospital (No. 1725). Code availability Not applicable Acknowledgments We thank the support from faculties and staff in the Maternal and Child Care Hospital of University of Ryukyus and all participants in this study. Funding Not applicable Availability of data and material Consent for publication Not applicable. 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J Perinatol 31:535–539. https://doi.org/10.1038/jp.2010.204 Tables Page 9/14 Table 1 Patient characteristics at birth. GA22–23-weeks (n = 10) GA24–25-weeks (n = 18) P-value Birth-weight (g) 539 ± 68 697 ± 155 0.003 Birth-weight (Z score) 0.13 ± 0.7 −0.38 ± 1.1 0.35 Head circumference at birth (cm) 20.4 ± 1.0 22.2 ± 1.4 0.002 Height at birth (cm) 29.3 ± 2.0 30.9 ± 4.0 0.027 SGA at birth No (%) 0 (0) 1 (5.6) 0.45a GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; SGA, small for gestational age. a Chi-square test GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; SGA, small for gestational age. a Chi square test Page 9/14 Page 9/14 Table 2 The progress of nutrition-related factors during hospitalization. GA22–23 weeks (n = 10) GA24–25- weeks (n = 18) P- value Days to achieved enteral feeds 100 ml/kg/day 13.0 ± 5.0 12.7 ± 3.7 0.94 Days to achieved enteral feeds 160 ml/kg/day 45.4 ± 22.5 23.7 ± 9.2 0.008 Head circumference at 37 weeks PMA (cm) 28.8 ± 1.2 30.4 ± 2.1 0.015 Head circumference at 37 weeks PMA (Z-score) −2.6 ± 0.8 −1.5 ± 1.4 0.016 Body weight at 37 weeks PMA (g) 1906 ± 321 2081 ± 379 0.17 Body weight at 37 weeks PMA (Z-score) −2.3 ± 1.0 −1.7 ± 1.3 0.16 No. of body weights less than 10% tile at 37 weeks PMA 9 11 0.10a Early postnatal weight loss (%) 10.4 ± 6.3 8.1 ± 6.3 0.37 The age of regain to birthweight 13.2 ± 5.9 11.8 ± 4.8 0.51 GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; PMA postmenstrual age. Values shown are mean ± SD. a Chi-square test Table 2 Table 2 The progress of nutrition-related factors during hospitalization. Page 10/14 Table 3 The short-term prognosis. GA22–23-weeks (n = 10) GA24–25-weeks (n = 18) P-valuea NEC No. (%) 1 (10) 0 (0) 0.17 PDA (indomethacin) No. (%) 8 (80) 9 (50) 0.13 PDA (surgery) No. (%) 3 (30) 3 (16.7) 0.41 ROP No. (%) 8 (80) 7 (38.9) 0.037 IVH grade Ⅲ–Ⅳ No. (%) 3 (30) 0 (0) 0.014 Home oxygen therapy No. (%) 3 (30) 10 (55.6) 0.40 Home HFNC No. (%) 1 (10) 1 (5.6) 0.28 Home CPAP No. (%) 0 (0) 0 (0) Tracheostomy No. (%) 0 (0) 2 (11.1) 0.27 Home feeding No. Tables (%) 1 (10) 1 (5.6) 0.66 GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; NEC, necrotizing enterocolitis; PDA, patent ductus arteriosus; ROP, retinopathy of prematurity; IVH intraventricular hemorrhage; HFNC, high flow nasal canula; CPAP, continuous positive airway pressure. a Chi-square test a Chi-square test Page 11/14 Page 11/14 Table 4 Complications associated with total parenteral nutrition. GA22–23-weeks GA24–25-weeks P-valuea Max BUN (mg/dl) 59.7 ± 16.6 (n = 10) 45.0 ± 10.8 (n = 18) 0.004 Max d-bil (mg/dl) 1.5 ± 0.7 (n = 10) 1.6 ± 0.8 (n = 18) 0.89 Max bile acid (µmol/L) 40.1 ± 20.2 (n = 10) 53.2 ± 23.0 (n = 18) 0.11 NH3 (µg/dl) 71.5 ± 7.4 (n = 4) 55.8 ± 18.3 (n = 4) 0.25 GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; BUN, blood urea nitrogen; d-bil, direct bilirubin. a Mann-Whitney U test GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; BUN, blood urea nitrogen; d-bil, direct bilirubin. Page 12/14 Table 5 The anthropometrics and developmental evaluations after discharge from the Neonatal Intensive Care Unit. GA22–23-weeks GA24–25-weeks P-value 1-year-old head circumference (cm) 43.0 ± 1.2 (n = 8) 43.7 ± 1.4 (n = 14) 0.13 1.5-year-old circumference (cm) 44.5 ± 0.9 (n = 6) 45.5 ± 1.7 (n = 10) 0.11 2-year-old DQ 71.3 ± 15.1 (n = 6) 78.1 ± 22.6 (n = 7) 0.20 3-year-old head circumference (cm) 48.9 ± 0.4 (n = 3) 48.2 ± 1.0 (n = 6) 0.12 3-year-old weight (kg) 11.4 ± 0.7 (n = 5) 12.6 ± 2.4 (n = 9) 0.26 3-year-old weight (Z score) −1.4 ± 0.5 (n = 5) −0.4 ± 1.6 (n = 9) 0.18 GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; DQ, developmental quotient. Table 5 GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; DQ, developmental quotient. GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; DQ, developmental quotient. GA22–23-weeks, 22–23 weeks gestational age; GA24–25-weeks, 24–25 weeks gestational age; DQ, developmental quotient. Figures Figures Page 13/14 Figure 1 Changes in the Z score of body weight during the course of hospitalization a) Mean Z score of body weight at 29 weeks PMA in the GA22–23-group: −2.3 ± 0.8 (n=10) Mean Z score of body weight at 29 weeks PMA in the GA24–25-group: −1.9 ± 0.6 (n=17) b) Mean Z score of body weight at 41 weeks PMA in the GA22–23-group: −1.0 ± 1.2 (n=8) Mean Z score of body weight at 41 weeks PMA in the GA24–25- group: −0.7 ± 1.2 (n=8) GA22–23-group, 22–23 weeks gestational age; GA24–25-group, 24–25 weeks gestational age; PMA, postmenstrual age. Page 14/14
https://openalex.org/W2993952021	https://europepmc.org/articles/pmc6898914?pdf=render	English	null	Knowledge and use of antibiotics in six ethnic groups: the HELIUS study	Antimicrobial resistance and infection control	2,019	cc-by	10,604	Abstract Background: The increase of antimicrobial resistance, mainly due to increased antibiotic use, is worrying. Preliminary evidence suggests that antibiotic use differs across ethnic groups in the Netherlands, with higher use in people of non-Dutch origin. We aimed to determine whether appropriate knowledge and use of antibiotics differ by ethnicity and whether knowledge on antibiotics is associated with antibiotic use. Methods: We performed a cross-sectional study analyzing baseline data (2011–2015) from a population-based cohort (HELIUS study), which were linked to data from a health insurance register. We included 21,617 HELIUS participants of South-Asian Surinamese, African-Surinamese, Turkish, Moroccan, Ghanaian, and Dutch origin. Fifteen thousand seven participants had available prescription data from the Achmea Health Data-base (AHD) in the year prior to their HELIUS study visit. Participants were asked five questions on antibiotic treatment during influenza-like illness, pneumonia, fever, sore throat and bronchitis, from which higher versus lower antibiotic knowledge level was determined. Number of antibiotic prescriptions in the year prior to the HELIUS study visit was used to determine antibiotic use. Results: The percentage of individuals with a higher level of antibiotic knowledge was lower among all ethnic minority groups (range 57 to 70%) compared to Dutch (80%). After correcting for baseline characteristics, including medical conditions, first-generation African Surinamese and Turkish migrants received a significantly lower number of antibiotic prescriptions compared to individuals of Dutch origin. Only second-generation Ghanaian participants received more prescriptions compared to Dutch participants (aIRR 2.09, 95%CI 1.06 to 4.12). Higher level of antibiotic knowledge was not significantly associated with the number of prescriptions (IRR 0.92, 95%CI 0.85 to 1.00). Conclusions: Levels of antibiotic knowledge varied between ethnic groups, but a lower level of antibiotic knowledge did not correspond with a higher number of antibiotic prescriptions. Keywords: Antibiotics, Antibiotic knowledge, Antibiotic use, Ethnic groups A recent meta-analysis showed a higher prevalence of antimicrobial resistance among migrants in Europe [2]. There is preliminary evidence in the Netherlands that the use of antibiotics also differs across ethnic groups, with a higher use of antibiotics among people of non- Dutch origin [3]. The reason for this difference, however, is unclear. It could be explained by increased incidence of bacterial infections, but, to the best of our knowledge, there is no evidence to support this hypothesis. Alterna- tively, knowledge about antibiotic use might vary across ethnic groups. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract As expectations and knowledge of the patient could potentially drive a physician’s decision to prescribe antibiotics, receiving prescriptions could also Knowledge and use of antibiotics in six ethnic groups: the HELIUS study Emelie C. Schuts1†, Eline van Dulm2† , Anders Boyd2,3, Marieke B. Snijder4,5, Suzanne E. Geerlings1, Maria Prins1,2 and Jan M. Prins1 Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 https://doi.org/10.1186/s13756-019-0636-x Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 https://doi.org/10.1186/s13756-019-0636-x Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 https://doi.org/10.1186/s13756-019-0636-x Open Access Open Access Background The emergence of antimicrobial resistance, along with the steady decline in antibiotic development, has been identified as a major health threat for the coming decade by the World Health Organization (WHO). Increase in antibiotic use is the main reason for this development [1] and as such, antibiotics should only be prescribed when there is a clear indication for use. Correspondence: evdulm@ggd.amsterdam.nl †Emelie C. Schuts and Eline van Dulm contributed equally to this work. 2Department of Infectious Diseases, Public Health Service Amsterdam, Nieuwe Achtergracht 100, 1018, WT, Amsterdam, The Netherlands Full list of author information is available at the end of the article Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 2 of 17 Page 2 of 17 differ between ethnic groups [4–6]. There are also cultural-specific approaches to dealing with authority, being the physician in this setting, which have explained differences in antibiotic use between countries [7]. Ethnicity was defined according to the country of birth of the participant as well as that of their parents [12]. Specifically, a participant is considered to be of non- Dutch ethnic origin if they fulfill either of the following criteria (i): they were born abroad and had at least one parent born abroad (first generation) or (ii) they were born in Netherlands but both their parents were born abroad (second generation). Dutch participants were born in the Netherlands and had both parents who were born in the Netherlands. After HELIUS data collection, the Surinamese group were further classi- fied according to self-reported ethnic origin (obtained by questionnaire), into ‘African Surinamese’, ‘South- Asian Surinamese’, ‘Javanese Surinamese’ and ‘other/ unknown Surinamese’. The HELIUS (Healthy life in an Urban Setting) study is a large-scale, population-based cohort study among different ethnic groups, which was established with the aim to investigate mechanisms underlying the impact of ethnicity on communicable and non-communicable dis- eases [8, 9]. In 2018, approximately 13% of the popula- tion of the Netherlands was of non-Western origin [10]. The largest non-Western population groups were individuals of Turkish (2.4%), Moroccan (2.3%) and Surinamese (2.0%) descent [10]. In Amsterdam, approxi- mately 36% of the population in 2018 was of non- Western descent [11]. The ethnic groups included in the HELIUS study are the largest ethnic minority groups of Amsterdam [9]. Amongst other data, data on antibiotic knowledge were collected. We were able to link these data at the individual level to data from a health insur- ance register on recent antibiotic use. Data linkage Permission to link participants’ individual data to outside health registries was asked in the written informed con- sent form [8]. Of the 22,165 HELIUS participants, 19, 895 agreed. HELIUS data of these individuals were linked to reimbursement data from the Achmea insur- ance company (Achmea Health Database, AHD) from 2010 until 2015. The AHD, obtained from the largest health insurance company in Amsterdam, contains all healthcare expenditures of every insured participant, including medications. A trusted third party linked data on reimbursed antibiotic prescriptions using an encrypted social security number and returned data without any identifying information. Procedures were in accordance with the General Data Protection Regulation [13]. This study then provides a unique opportunity to de- termine whether knowledge about and use of antibiotics vary between ethnic groups, and if so, whether differ- ences in antibiotic use can be attributed to differences in knowledge about antibiotics. We hypothesized that antibiotic use differs among ethnic groups as a result of differences in knowledge. Study population and design y p p g The HEalthy LIfe in an Urban Setting (HELIUS) study is a multiethnic cohort study conducted in Amsterdam, which focuses on cardiovascular disease (e.g. diabetes), mental health (e.g. depressive disorders), and infectious diseases [8, 9]. In brief, baseline data collection took place in 2011–2015 and included people aged 18 to 70 years of Dutch, Surinamese, Ghanaian, Moroccan, and Turkish origin. A random sample of participants, strati- fied by ethnic origin, was taken from the municipality register of Amsterdam. Participants filled in an extensive self-administered questionnaire (variables included in the questionnaire are described elsewhere) [9] and underwent a physical examination during which biological samples were obtained [9]. No information was provided regarding appropriate antibiotic use. Between 2011 and 2015, 24,789 persons were included. Data collection procedures have been previously described in detail [9]. Both questionnaire data and physical examination data were available for 22, 165 participants. The HELIUS study was conducted in accordance with the Declaration of Helsinki and was ap- proved by the AMC Ethical Review Board. All participants provided written informed consent. Inclusion and exclusion criteria for present study Of the 22,165 participants, we excluded those of Javan- ese Surinamese or other/unknown Surinamese origin and those with another/unknown ethnic origin because of small participant numbers. For analyses on antibiotic use, we included those who gave permission for data linkage and could be linked to the AHD. To reduce bias for individuals with short-term insurance, we excluded those who were insured with Achmea for less than 365 days in the year preceding their HELIUS study visit. Outcome variables The primary outcomes were level of antibiotic know- ledge and antibiotic use during the year prior to the HELIUS visit. Level of antibiotic knowledge was based on five questions, used in other studies [4, 6, 14], which asked the perceived necessity (yes/no) for antibiotic treatment during influenza-like illness, pneumonia, fever, sore throat and bronchitis. Using these questions, we created an overall knowledge score of antibiotic use by summing the total number of correct responses, Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 3 of 17 Page 3 of 17 Analysis on antibiotic use in the year prior to HELIUS study visit included all HELIUS participants who were linked to the AHD and were insured for at least 365 days with Achmea in the year prior to their HELIUS study visit. Determinants for having received ≥1 antibiotic pre- scription were assessed using logistic regression. The same multivariable approach as above was used for this outcome. We also compared antibiotic use during the entire period insured at Achmea versus the year prior to HELIUS study visit to assess differences when consider- ing longer time periods. resulting in a score ranging from 0 to 5. A two- parameter logistic regression model was fitted to the five binary items based on the assumptions of item response theory (see Additional file 1). From this model, “higher” and “lower” levels of antibiotic knowledge were defined by a knowledge score of ≥4 and < 4, respectively. resulting in a score ranging from 0 to 5. A two- parameter logistic regression model was fitted to the five binary items based on the assumptions of item response theory (see Additional file 1). From this model, “higher” and “lower” levels of antibiotic knowledge were defined by a knowledge score of ≥4 and < 4, respectively. y g p y Antibiotic use was obtained from linked AHD data and was based on the total number of reimbursed antibi- otics (classified by ATC code J01; anti-infectives for sys- temic use) dispensed by community pharmacies from 2010 until 2015. We evaluated antibiotic use (yes/no) in the year prior to the HELIUS study visit, as well as the number of antibiotic prescriptions over the past year and during the entire insured period. Statistical analyses Multicollinearity was verified using variance inflation factors, while any variable with an inflation factor of ≥4 was considered multicollinear and excluded from the model. To understand whether the association between ethnicity and outcome was modified by demographic variables, interaction between ethnicity and other demo- graphic variables was also assessed in all multivariable models. Sociodemographics, health status, antibiotic knowledge level and questions on antibiotic use were presented by ethnicity. To assess selection bias resulting from AHD data linkage, the same variables were compared between participants who were successfully versus unsuccessfully linked. Comparisons between ethnic groups were made using a Pearson’s χ2 or Fisher exact test for categorical data and Kruskal-Wallis rank test for continuous variables. The three variables involving antibiotic-related behav- ior were not initially considered in the final multivariable models. To assess whether ethnic differences in anti- biotic use could be explained by patterns of antibiotic- related behavior, additional multivariable models includ- ing these variables were constructed for the endpoints (i) having received ≥1 antibiotic prescription and (ii) total number of antibiotic prescriptions. Analysis on level of antibiotic knowledge included all HELIUS participants with available data. Odds ratios (OR) comparing levels of antibiotic knowledge across determinants and their 95% confidence intervals (CI) were estimated using logistic regression. All variables with an associated p-value < 0.2 in univariable analyses were included in a full multivariable model and variables with a p-value above this level were removed in backwards-stepwise fashion. Given that the research aim was to determine differences between ethnicity, ethnic groups were forced in all models. This multivariable ap- proach was chosen to not only assess other variables as- sociated with antibiotic knowledge, but also to understand the extent of confounding bias when asses- sing the relationship between ethnicity and outcome variables. Figure 1 provides an overview of all descriptive ana- lysis and modeling used in the study. Significance was determined using a p-value < 0.05. All analyses were conducted with Stata 13.1 (StataCorp., College Station, Texas, USA). Outcome variables Determinants for the total number of antibiotic pre- scriptions were then evaluated. As this outcome con- tained a high proportion of zero values and was over- dispersed, we used a zero-inflated negative binomial re- gression model. This model contains two parts: one ac- counting for zero values in the count distribution (zero- inflated) and another accounting for the over-dispersed count distribution (negative binomial). Covariates for the zero-inflated part were determined a priori from the risk-factor analysis on ≥1 antibiotic prescription. Covari- ates for the negative binomial part were selected from covariates with a p-value < 0.2 in univariable analyses and variables above this p-value were removed in backwards-stepwise fashion. Incidence risk ratios (IRR) comparing the number of antibiotics prescribed over the past year across levels of determinants were estimated from this model. Other variables Independent variables were obtained from the HELIUS study questionnaire (migration generation; sex; age; level of education; marital status; self-reported medical condi- tions; smoking; alcohol consumption; difficulty with the Dutch language and perceived health) and physical examination (body mass index (BMI, kg/m2)). Variables on antibiotic-related behavior were: not having finished antibiotic treatment; having saved antibiotics for later; and ever having asked the general practitioner (GP) for antibiotics. Definitions and grouping of variables are extensively described elsewhere [8]. Participants Of the 22,165 HELIUS participants with available data, 21,617 were eligible after applying exclusion criteria. Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 4 of 17 Fig. 1 Overview of descriptive analysis and models used in the study. Abbreviations: HELIUS – Healthy Life in an Urban Setting; AHD – Achmea Health Database Fig. 1 Overview of descriptive analysis and models used in the study. Abbreviations: HELIUS – Healthy Life in an Urban Setting; AHD – Achmea Health Database ptive analysis and models used in the study. Abbreviations: HELIUS – Healthy Life in an Urban Setting; AHD – Achmea Fig. 1 Overview of descriptive analysis and models used in the study. Abbreviations: HELIUS – Healthy Life in an Urban Setting; AHD – Achmea Health Database Their baseline characteristics, stratified by ethnicity, are shown in Table 1. Median age of participants was 46 years (IQR 34 to 55) and 58% were women. The propor- tion of several medical conditions predisposing individ- uals to antibiotic treatment differed by ethnicity. Of these conditions, South-Asian Surinamese participants had the highest prevalence of self-reported diabetes mel- litus (17%) and cerebrovascular accident (CVA) (6%) over the last 12 months. Turkish individuals had more prevalent artery stenosis (10%), severe or chronic fatigue (45%) and respiratory diseases (15%), whereas Ghanaians more frequently reported high blood pressure (33%). Excellent perceived health was reported in 12% of Dutch participants in contrast to 3.3% of Turkish participants. their GP for antibiotics ranged from 0.6% in African Surinamese participants to 1.9% in Turkish and Moroc- can participants. As shown in Table 2, there was a significantly lower odds of individuals with higher level of antibiotic know- ledge among all non-Dutch ethnic groups compared to Dutch individuals (overall p < 0.001) (Table 2). Across all non-Dutch groups, second-generation participants had a higher level of antibiotic knowledge than first-generation participants; however, results remained significantly lower compared to the Dutch group. In multivariable analysis, all ethnic minority groups had lower odds for higher level of antibiotic knowledge compared to Dutch (overall p < 0.001), although the ef- fect for second-generation Ghanaian participants was not statistically significant. The odds for higher level of antibiotic knowledge were higher in all age groups > 25 years of age (except for those ≥65) when compared to ≤25 years of age. Participants Furthermore, women had a significantly higher odds of having a higher level of antibiotic know- ledge compared to males. Lower odds for a higher level of antibiotic knowledge were found for the following medical conditions: myocardial Infarction (MI), severe or chronic fatigue, respiratory diseases and having a BMI ≥25. Lower odds for higher level of antibiotic knowledge were also seen among individuals who regularly or occa- sionally requested antibiotics from their GP or who regularly or occasionally did not finish treatment. Ethnic differences in antibiotic knowledge In several ethnic groups, there were substantial propor- tions of individuals reporting the need to be treated with antibiotics for illnesses without indication, as shown in Table 1. The number of people reporting to have been treated with antibiotics and not having regularly com- pleted their antibiotic treatment was low across all eth- nic groups, ranging from 0.1% in Dutch participants to 2.1% in Ghanaian participants. Few individuals regularly saved their antibiotics for later use, ranging from < 0.1% in Dutch participants to 0.3% in Turkish participants. The percentage of participants having regularly asked Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 5 of 17 Table 1 Characteristics of the HELIUS study population (N = 21,617) by ethnicity Variablesa Ethnicity Dutch (N = 4564) South-Asian Surinamese (N = 3043) African Surinamese (N = 4151) Ghanaian (N = 2339) Turkish (N = 3614) Moroccan (N = 3906) Sociodemographics Female sex 2475 54% 1672 55% 2535 61% 1434 61% 1980 55% 2392 61% Age in years, median (IQR) 47 (34–58) 48 (35–56) 50 (40–57) 47 (38–53) 42 (31–50) 40 (30–50) Migration generation 1st generation N.A. N.A. 2328 77% 3468 84% 2231 95% 2544 70% 2680 69% 2nd generation N.A. N.A. 715 23% 683 16% 108 4.6% 1080 30% 1226 31% Educational level Unknown 25 0.6% 16 0.5% 36 0.9% 42 1.8% 38 1.1% 38 1.0% No school/elementary school 150 3.3% 437 14% 231 6% 660 28% 1135 31% 1205 31% Lower vocational/lower secondary school 646 14% 1010 33% 1477 36% 917 39% 889 25% 694 18% Intermediate vocational/ intermediate secondary school 994 22% 885 29% 1464 35% 578 25% 1020 28% 1294 33% Higher vocational/university 2749 60% 695 23% 943 23% 142 6% 532 15% 675 17% Marital status Married/registered partnership 1724 38% 1043 34% 766 19% 420 18% 2208 61% 2285 59% Cohabiting 914 20% 311 10% 441 11% 427 19% 132 3.7% 110 2.8% Unmarried/never married 1474 32% 1001 33% 2231 54% 779 34% 761 21% 1010 26% Divorced/separated 356 8% 580 19% 617 15% 656 28% 407 11% 414 11% Widow/widower 87 1.9% 92 3.0% 65 1.6% 23 1.0% 90 2.5% 69 1.8% Health status Self-reported medical conditions (previous 12 months) Diabetes mellitus 102 2.2% 521 17% 419 10% 185 8% 336 9% 389 10% CVA/one-sided loss of bodily function ≤1 day 160 3.5% 212 7% 261 6% 95 4.1% 196 5% 195 5% MI incl. Ethnic differences in antibiotic knowledge ≥half hour chest pain or dotter/bypass operation 233 5% 491 16% 440 11% 225 10% 591 16% 476 12% Severe heart condition 67 1.5% 120 4.0% 105 2.5% 75 3.2% 153 4.3% 58 1.5% Malignant disorder 103 2.3% 70 2.3% 85 2.1% 33 1.4% 73 2.0% 46 1.2% Severe or chronic fatigue 633 14% 1032 34% 956 23% 186 8% 1602 45% 1465 38% High blood pressure 534 12% 720 24% 1230 30% 770 33% 610 17% 546 14% Artery stenosis 85 1.9% 193 6% 181 4.4% 115 5% 348 10% 200 5% Respiratory diseases 345 8% 433 14% 354 9% 117 5% 556 15% 446 11% Serious/persistent intestinal disorders 249 5% 248 8% 308 7% 70 3.0% 433 12% 391 10% Psoriasis 136 3.0% 168 6% 128 3.1% 71 3.1% 154 4.3% 121 3.1% (Chronic) eczema 420 9% 406 13% 370 9% 71 3.1% 471 13% 423 11% Incontinence 309 7% 326 11% 342 8% 108 4.7% 464 13% 300 8% Body Mass Index (kg/m2), median (IQR) 24.1 (21.9– 26.7) 25.7 (23.2– 28.8) 27.0 (23.9– 30.8) 27.9 (25.0– 31.2) 27.9 (24.6– 31.7) 27.0 (23.9– 30.7) Smoking Yes 1129 25% 861 28% 1309 32% 104 4.5% 1240 35% 525 13% No, never 1689 37% 1758 58% 2016 49% 2027 87% 1700 47% 2874 74% No, but ever 1737 38% 413 14% 805 19% 191 8% 648 18% 492 13% Alcohol consumption Never 297 7% 1072 35% 1002 24% 806 35% 2414 67% 3265 84% Table 1 Characteristics of the HELIUS study population (N = 21,617) by ethnicity Schuts et al. Ethnic differences in antibiotic knowledge Antimicrobial Resistance and Infection Control (2019) 8:200 Page 6 of 17 Table 1 Characteristics of the HELIUS study population (N = 21,617) by ethnicity (Continued) Table 1 Characteristics of the HELIUS study population (N = 21,617) by ethnicity (Continued) Variablesa Ethnicity Dutch (N = 4564) South-Asian Surinamese (N = 3043) African Surinamese (N = 4151) Ghanaian (N = 2339) Turkish (N = 3614) Moroccan (N = 3906) Not in previous 12 months 110 2.4% 251 8% 292 7% 408 18% 358 10% 338 9% Monthly or less 436 10% 758 25% 1242 30% 508 22% 367 10% 127 3.3% 2–4 times per month 894 20% 541 18% 873 21% 291 13% 257 7% 87 2.2% 2–3 times per week 1413 31% 262 9% 439 11% 193 8% 132 3.7% 56 1.4% ≥4 times per week 1408 31% 147 5% 272 7% 109 4.7% 57 1.6% 16 0.4% Difficulty with Dutch language Yes N.A. N.A. Ethnic differences in antibiotic knowledge 711 23% 520 13% 1926 83% 2136 60% 1774 46% Perceived health Excellent 541 12% 162 5% 303 7% 226 10% 117 3.3% 166 4.3% Very good 1381 30% 310 10% 571 14% 458 20% 383 11% 384 10% Good 2205 48% 1623 53% 2335 56% 1180 51% 1871 52% 1871 48% Mediocre 402 9% 811 27% 834 20% 383 16% 921 26% 1223 31% Bad 28 0.6% 131 4.3% 101 2.4% 88 3.8% 307 9% 241 6% Antibiotics Knowledge concerning antibioticsb Antibiotics effective for influenza 324 7% 554 19% 592 15% 658 29% 744 21% 648 18% Antibiotics effective for pneumonia 4166 92% 2304 77% 3114 77% 1312 58% 2587 73% 2741 73% Antibiotics effective for feverc 689 15% 552 19% 679 17% 586 26% 898 26% 614 17% Antibiotics effective for sore throatc 672 15% 760 26% 1089 27% 720 32% 1203 34% 978 26% Antibiotics effective for bronchitis 2246 50% 1385 50% 1862 46% 919 41% 1235 35% 1485 40% Higher level of antibiotic knowledged 3638 80% 1996 68% 2737 69% 1248 57% 2128 62% 2528 70% Did not finish antibiotic treatment Yes, regularly 5 0.1% 49 1.6% 44 1.1% 48 2.1% 41 1.2% 44 1.1% Yes, occasionally 332 7% 312 10% 527 13% 174 8% 424 12% 445 12% Always finished or no antibiotics 4203 93% 2646 88% 3524 86% 2053 90% 3104 87% 3361 87% Saved antibiotics for later Yes, regularly 2 0.0% 7 0.2% 9 0.2% 5 0.2% 10 0.3% 6 0.2% Yes, occasionally 37 0.8% 45 1.5% 68 1.7% 62 2.7% 60 1.7% 46 1.2% No, never 297 7% 304 10% 492 12% 146 6% 387 11% 430 11% Not applicable (no antibiotics) 4203 93% 2646 88% 3524 86% 2053 91% 3104 87% 3361 87% Ever asked GP for antibiotics Yes, regularly 38 0.8% 34 1.1% 26 0.6% 36 1.6% 67 1.9% 71 1.9% Yes, occasionally 824 18% 491 16% 607 15% 401 18% 734 21% 634 17% No, never 3674 81% 2482 83% 3441 84% 1835 81% 2744 77% 3074 81% Missing data, n: marital status 128; diabetes 78; stroke 55; myocardial infarction 33; heart condition 83; malignant disorders 137; fatigue 145; high blood pressure 101; artery stenosis 140; respiratory diseases 115; bowel diseases 115; psoriasis 98; eczema 117; incontinence 126; BMI 23; smoking 107; alcohol 127; perceived health 61; AB effective for influenza 614; AB effective for pneumonia 477; AB effective for fever 685; AB effective for sore throat 625; AB effective for bronchitis 663; asked GP for AB 414; did not finish treatment 292; saved AB 325 Abbreviations: IQR Inter Quartile Range, CVA Cerebro Vascular Accident, MI Myocardial infarction, N.A. Ethnic differences in antibiotic knowledge Not Applicable, GP General Practitioner N.A. Not applicable (categories not applicable due to Dutch ethnicity) aAll variables are reported as n (%), unless otherwise indicated bAnswered “yes” to the statements below cThe Dutch General Practitioners guidelines (and those of other European countries) advise against the use of antibiotics for fever in general or sore throat, as they usually constitute viral infections, with only a few exceptions in both cases. Therefore, antibiotics are in general not appropriate for dBased on a summed score with cutoff determined by an Item Response Theory model (≥4 out of 5 antibiotic knowledge questions correctly answered was considered as having a higher level of knowledge) Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 7 of 17 Table 2 Variables associated with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) Univariable Multivariable (N = 20,081a)# OR (95% CI) P-values aOR (95% CI) P-values Sociodemographics Ethnicity <.001 <.001 Dutch Ref Ref South-Asian Surinamese 1st generation 0.49 0.44–0.55 0.53 0.47–0.60 2nd generation 0.56 0.47–0.67 0.60 0.50–0.73 African Surinamese 1st generation 0.51 0.46–0.57 0.53 0.47–0.59 2nd generation 0.75 0.62–0.91 0.79 0.64–0.96 Ghanaian 1st generation 0.31 0.27–0.34 0.31 0.27–0.35 2nd generation 0.64 0.41–0.98 0.74 0.47–1.18 Turkish 1st generation 0.35 0.31–0.39 0.40 0.36–0.45 2nd generation 0.56 0.48–0.65 0.62 0.53–0.74 Moroccan 1st generation 0.51 0.45–0.57 0.56 0.50–0.63 2nd generation 0.71 0.61–0.83 0.75 0.63–0.89 Female sex 1.18 1.11–1.25 <.001 1.32 1.23–1.40 <.001 Age <.001 <.001 < 25 years Ref Ref 25–34 years 1.24 1.01–1.34 1.32 1.16–1.50 35–44 years 0.99 0.81–1.05 1.30 1.14–1.49 45–54 years 0.85 0.71–0.91 1.19 1.04–1.37 55–64 years 0.95 0.78–1.02 1.26 1.08–1.45 ≥65 years 1.06 0.84–1.22 1.15 0.95–1.39 Educational level <.001 Unknown Ref No school/elementary school 1.10 0.79–1.55 Lower vocational/lower secondary school 1.27 0.91–1.77 Intermediate vocational/ intermediate secondary school 1.54 1.11–2.15 Higher vocational/university 2.06 1.47–2.87 Marital status <.001 Married/registered partnership Ref Cohabiting 1.15 1.04–1.27 Unmarried/never married 1.08 1.01–1.16 Divorced/separated 0.81 0.74–0.89 Widow/widower 1.10 0.88–1.36 Health status Self-reported medical conditions (previous 12 months) Diabetes mellitus 0.70 0.63–0.77 <.001 CVA/one-sided loss of bodily function ≤1 day 0.85 0.75–0.97 .015 MI incl. ≥half hour chest pain or dotter/bypass operation 0.71 0.65–0.77 <.001 0.89 0.81–0.98 .017 Schuts et al. Ethnic difference in antibiotic use Of the 19,895 HELIUS participants consenting to link their data to other health registries, 15,461 were linked to the AHD (77.7%). Of these 15,461 participants, 15,007 (97%) were insured for ≥365 days in the year prior to their HELIUS study visit. Additional file 2: Table S1 shows the characteristics of the study participants linked versus not linked to the AHD. Participants present in the AHD register had a lower level of education, higher prevalence of medical conditions, and less often had higher levels of antibiotic knowledge. Table 5 shows the results from the analysis on the as- sociation between ethnicity and total number of anti- biotic prescriptions received in the year prior to the HELIUS study visit. Differences across ethnic groups were observed overall for the number of antibiotic pre- scriptions in both univariable and multivariable analysis (both p = 0.004). First-generation African Surinamese and Turkish migrants had a significantly lower number of antibiotic prescriptions compared to individuals of Dutch origin. Only second-generation Ghanaian partici- pants has more prescriptions compared to Dutch partici- pants. Furthermore, female sex, diabetes mellitus, MI, malignant disorder, respiratory disease, eczema and worse perceived health were significantly associated with a higher number of antibiotic prescriptions. Table 3 describes antibiotic use according to ethnicity for participants registered in the AHD. In total, 31,530 antibiotic prescriptions were recorded over the study period. The proportion of participants receiving ≥1 anti- biotic prescription in the year prior to their HELIUS study visit was highest among first-generation Turkish participants (25%) and was comparably high among second-generation Ghanaian and first-generation Moroccan participants (both 25%). The proportion of participants receiving ≥1 antibiotic prescription in the year prior to the HELIUS study visit was lowest in Dutch and second generation South-Asian Surinamese partici- pants (both 16%). Having a higher level of antibiotic knowledge was not significantly associated with the number of prescriptions when included in multivariable analysis (p = 0.446). No significant interactions between ethnicity and sex or education were observed. Finally, adjusting the associ- ation between ethnicity and antibiotic use for antibiotic- related behaviors did not change these associations. When considering the entire period during which par- ticipants were insured at Achmea prior to the HELIUS study visit (median 6.0 years, IQR 5.0 to 6.0), the propor- tion of participants receiving ≥1 antibiotic prescription was highest among first generation Turkish participants (69%) and lowest in second-generation Ghanaian partici- pants (49%). Ethnic differences in antibiotic knowledge Antimicrobial Resistance and Infection Control (2019) 8:200 Page 9 of 17 Table 2 Variables associated with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) (Continued) Univariable Multivariable (N = 20,081a)# OR (95% CI) P-values aOR (95% CI) P-values Did not finish treatment <.001 <.001 Always finished or no antibiotics Ref Ref Yes, regularly 0.51 0.39–0.67 0.71 0.54–0.94 Yes, occasionally 0.73 0.66–0.80 0.80 0.73–0.88 Abbreviations: OR Odds Ratio, aOR adjusted Odds Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner aFewer observations in the multivariable model than in the total study population were due to missing observations on certain covariates #We found significant interactions between ethnicity and sex (p = 0.007) and ethnicity and age (p = 0.047) Table 2 Variables associated with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) (Continued) Univariable Multivariable (N = 20,081a)# OR (95% CI) P-values aOR (95% CI) P-values Did not finish treatment <.001 <.001 Always finished or no antibiotics Ref Ref Yes, regularly 0.51 0.39–0.67 0.71 0.54–0.94 Yes, occasionally 0.73 0.66–0.80 0.80 0.73–0.88 Abbreviations: OR Odds Ratio, aOR adjusted Odds Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner aFewer observations in the multivariable model than in the total study population were due to missing observations on certain covariates #We found significant interactions between ethnicity and sex (p = 0.007) and ethnicity and age (p = 0.047) Table 2 Variables associated with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) th higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) Abbreviations: OR Odds Ratio, aOR adjusted Odds Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner aFewer observations in the multivariable model than in the total study population were due to missing observations on certain covariates #We found significant interactions between ethnicity and sex (p = 0.007) and ethnicity and age (p = 0.047) Abbreviations: OR Odds Ratio, aOR adjusted Odds Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner aFewer observations in the multivariable model than in the total study population were due to missing observations on certain covariates #We found significant interactions between ethnicity and sex (p = 0.007) and ethnicity and age (p = 0.047) (p < 0.001) and multivariable analysis (p < 0.001). Discussion Our study shows that knowledge on the need to use an- tibiotics for treatment is lower among all ethnic minority groups compared to Dutch, with second generation eth- nic minorities showing higher levels of knowledge com- pared to first generation migrants. We also observed ethnic differences in the use of antibiotics, with a higher proportion having received at least one prescription, but a lower mean number of antibiotic prescriptions among some ethnic minority groups compared to Dutch. The only ethnic group with a significantly higher number of Ethnic difference in antibiotic use The mean number of prescriptions during the entire insured period was comparable to the mean number of prescriptions in the year prior to HELIUS study visit for all ethnic groups (Table 2). Ethnic differences in antibiotic knowledge In mul- tivariable analysis, compared to Dutch individuals, first and second generation Ghanaian individuals and first- generation Moroccan individuals had significantly higher odds of receiving ≥1 antibiotic prescription. Adding vari- ables on antibiotic-related behavior and level of anti- biotic use knowledge to the multivariable model did not change these associations. Ethnic differences in antibiotic knowledge Antimicrobial Resistance and Infection Control (2019) 8:200 Page 9 of 17 Schuts et al. Ethnic differences in antibiotic knowledge Antimicrobial Resistance and Infection Control (2019) 8:200 Page 8 of 17 Table 2 Variables associated with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) (Continued) Univariable Multivariable (N = 20,081a)# OR (95% CI) P-values aOR (95% CI) P-values Severe heart condition 0.61 0.52–0.73 <.001 Malignant disorder 0.97 0.78–1.20 .752 Severe or chronic fatigue 0.80 0.75–0.85 <.001 0.89 0.82–0.95 .001 High blood pressure 0.78 0.73–0.84 <.001 Artery stenosis 0.69 0.61–0.78 <.001 Respiratory diseases 0.68 0.62–0.75 <.001 0.80 0.72–0.88 <.001 Serious/persistent intestinal disorders 0.88 0.79–0.98 .024 Psoriasis 0.76 0.66–0.89 .001 (Chronic) eczema 0.93 0.84–1.02 .121 Incontinence 0.84 0.76–0.93 .001 Body Mass Index <.001 .001 < 18.5 Ref Ref 18.5–25 1.03 0.81–1.31 1.02 0.80–1.31 25–30 0.80 0.63–1.01 0.96 0.75–1.24 30–40 0.67 0.52–0.85 0.86 0.66–1.11 ≥40 0.58 0.43–0.78 0.78 0.57–1.08 Smoking <.001 Yes Ref No, never 1.03 0.96–1.11 No, but ever 1.19 1.09–1.30 Alcohol usage <.001 Never Ref Not in previous 12 months 0.89 0.80–1.00 Monthly or less 1.10 1.01–1.20 2–4 times per month 1.27 1.16–1.40 2–3 times per week 1.35 1.22–1.49 ≥4 times per week 1.59 1.42–1.78 Difficulty with Dutch language <.001 No Ref Yes 0.65 0.61–0.69 Not applicable 1.70 1.56–1.85 Perceived health <.001 Excellent Ref Very good 0.97 0.85–1.12 Good 0.83 0.73–0.93 Mediocre 0.63 0.56–0.72 Bad 0.48 0.40–0.57 Antibiotics Ever asked GP for antibiotics <.001 <.001 N R f R f Table 2 Variables associated with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) with higher antibiotic knowledge in HELIUS study population (N = 21,617) (logistic regression analysis) edge in HELIUS study population (N = 21,617) (logistic regression analysis) Univariable Multivariable (N = 20,081a)# OR (95% CI) P-values aOR (95% CI) P-values 0.61 0.52–0.73 <.001 0.97 0.78–1.20 .752 0.80 0.75–0.85 <.001 0.89 0.82–0.95 .001 0.78 0.73–0.84 <.001 0.69 0.61–0.78 <.001 0.68 0.62–0.75 <.001 0.80 0.72–0.88 <.001 0.88 0.79–0.98 .024 0.76 0.66–0.89 .001 0.93 0.84–1.02 .121 0.84 0.76–0.93 .001 <.001 .001 Ref Ref 1.03 0.81–1.31 1.02 0.80–1.31 0.80 0.63–1.01 0.96 0.75–1.24 0.67 0.52–0.85 0.86 0.66–1.11 0.58 0.43–0.78 0.78 0.57–1.08 <.001 Ref 1.03 0.96–1.11 1.19 1.09–1.30 <.001 Ref 0.89 0.80–1.00 1.10 1.01–1.20 1.27 1.16–1.40 1.35 1.22–1.49 1.59 1.42–1.78 <.001 Ref 0.65 0.61–0.69 1.70 1.56–1.85 <.001 Ref 0.97 0.85–1.12 0.83 0.73–0.93 0.63 0.56–0.72 0.48 0.40–0.57 <.001 <.001 Ref Ref 0.51 0.40–0.65 0.60 0.46–0.77 0 57 0 53 0 61 0 59 0 55 0 64 Yes, occasionally Schuts et al. Determinants of antibiotic use and number of prescriptions Table 4 shows the results from the analysis on the asso- ciation between ethnicity and having received ≥1 anti- biotic prescription in the year prior to the HELIUS study visit. Differences across ethnic groups were observed overall for any antibiotic prescription in both univariable Page 10 of 17 Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Table 3 Antibiotic use in participants linked to AHD (N = 15,007) stratified by ethnicity Ethnicity Dutch South-Asian Surinamese African Surinamese Ghanaian Turkish Moroccan (N = 2071) 1st gen (N = 1645) 2nd gen (N = 452) 1st gen (N = 2334) 2nd gen (N = 432) 1st gen (N = 1789) 2nd gen (N = 84) 1st gen (N = 2102) 2nd gen (N = 776) 1st gen (N = 2119) 2nd gen (N = 857) Duration of insurance at Achmea (in years) between 2010 and 2015, median (IQR) 6.0 (4.0–6.0) 6.0 (6.0–6.0) 6.0 (4.0–6.0) 6.0 (6.0–6.0) 6.0 (5.0–6.0) 6.0 (6.0–6.0) 6.0 (4.0–6.0) 6.0 (96.0–6.0) 6.0 (5.0–6.0) 6.0 (6.0–6.0) 6.0 (4.0–6.0) Within year prior to HELIUS study visit Participants with ≥1 ABP 16% 22% 16% 17% 17% 22% 25% 25% 19% 25% 17% Number of ABP among all participants included in the AHD Mean 0.26 0.39 0.26 0.28 0.28 0.33 0.55 0.40 0.34 0.41 0.28 Median (IQR) 0.00 (0.0–0.0) 0.00 (0.0–0.0) 0.00 (0.0–0.0) 0.00 (0.0–0.0) 0.00 (0.0–0.0) 0.00 (0.0–0.0) 0.00 (0.0–0.5) 0.00 (0.0–1.0) 0.00 (0.0–0.0) 0.00 (0.0–1.0) 0.00 (0.0–0.0) Number of ABP among participants with ≥1 ABP Mean 1.66 1.75 1.59 1.58 1.64 1.51 2.19 1.57 1.78 1.64 1.63 Median (IQR) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 2.00 (1.0–3.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) 1.00 (1.0–2.0) During entire insured period Participants with ≥1 ABP 51% 63% 53% 56% 51% 62% 49% 69% 59% 67% 54% Number of ABP per year among all participants included in the AHD Mean 0.31 0.46 0.30 0.31 0.30 0.35 0.34 0.44 0.38 0.43 0.33 Median (IQR) 0.17 (0.0–0.3) 0.17 (0. 0–0.6) 0.17 (0. 0–0.3) 0.17 (0.0–0.3) 0.17 (0.0–0.3) 0.17 (0.0–0.5) 0.00 (0.0–0.7) 0.25 (0.0–0.7) 0.17 (0.0–0.5) 0.17 (0.0–0.6) 0.17 (0.0–0.5) Abbreviations: ABP Antibiotic Prescription, IQR Inter Quartile Range Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 11 of 17 Schuts et al. Determinants of antibiotic use and number of prescriptions Antimicrobial Resistance and Infection Control (2019) 8:200 Page 12 of 17 antibiotic prescription in the year prior to HELIUS visit in participants linked to ed) Univariable Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior OR (95% CI) P-values aOR (95% CI) P-values aOR (95% CI) P-values 1.38 1.18–1.62 <.001 1.78 1.60–1.98 <.001 1.28 1.13–1.45 <.001 1.24 1.09–1.40 .001 1.45 1.18–1.79 .001 1.93 1.52–2.47 <.001 1.33 1.02–1.74 .037 1.86 1.71–2.02 <.001 1.20 1.08–1.33 .001 1.16 1.04–1.29 .008 1.36 1.25–1.49 <.001 1.46 1.25–1.70 <.001 0.80 0.67–0.96 .014 0.77 0.64–0.92 .004 2.19 1.96–2.44 <.001 1.66 1.47–1.87 <.001 1.59 1.41–1.81 <.001 1.87 1.64–2.12 <.001 1.24 1.07–1.43 .004 1.22 1.05–1.41 .009 1.28 1.05–1.56 .015 1.30 1.15–1.47 <.001 2.08 1.85–2.35 <.001 1.32 1.15–1.52 <.001 1.32 1.15–1.52 <.001 <.001 Ref 1.03 0.72–1.46 1.24 0.87–1.76 1.64 1.15–2.33 1.97 1.31–2.97 .184 <.001 .003 Ref Ref Ref 0.99 0.90–1.09 0.78 0.69–0.87 0.82 0.72–0.92 0.90 0.80–1.02 0.91 0.79–1.04 0.93 0.81–1.07 <.001 .017 .012 Ref Ref Ref 0.88 0.77–1.02 0.96 0.82–1.12 0.95 0.81–1.12 0.68 0.61–0.77 0.83 0.72–0.95 0.82 0.71–0.94 0.73 0.64–0.83 0.93 0.79–1.09 0.92 0.78–1.08 0.64 0.55–0.75 0.92 0.76–1.10 0.89 0.74–1.08 0.48 0.39–0.58 0.70 0.55–0.88 0.68 0.54–0.86 <.001 Ref 1.32 1.21–1.43 0.80 0.71–0.91 <.001 <.001 .002 Ref Ref Ref 1.09 0.86–1.37 1.05 0.83–1.34 1.03 0.80–1.31 1.55 1.27–1.90 1.22 0.99–1.51 1.20 0.97–1.49 2.47 2.01–3.04 1.37 1.09–1.72 1.32 1.05–1.67 3.85 3.02–4.91 1.69 1.28–2.23 1.59 1.20–2.11 <.001 Table 4 Variables associated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to AHD (N = 15,007) (logistic regression analysis) (Continued) Univariable Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior OR (95% CI) P-values aOR (95% CI) P-values aOR (95% CI) P-values CVA/one-sided loss of bodily function ≤1 day 1.38 1.18–1.62 <.001 MI incl. Determinants of antibiotic use and number of prescriptions Antimicrobial Resistance and Infection Control (2019) 8:200 Page 11 of 17 Table 4 Variables associated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to AHD (N = 15,007) (logistic regression analysis) Univariable Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior OR (95% CI) P-values aOR (95% CI) P-values aOR (95% CI) P-values Sociodemographics Ethnicity <.001 <.001 .004 Dutch Ref Ref Ref South-Asian Surinamese 1st generation 1.57 1.33–1.85 1.05 0.86–1.27 1.04 0.85–1.26 2nd generation 1.05 0.79–1.38 0.95 0.71–1.28 0.92 0.68–1.24 African Surinamese 1st generation 1.15 0.98–1.35 0.89 0.75–1.07 0.88 0.73–1.05 2nd generation 1.14 0.87–1.50 1.02 0.76–1.36 0.96 0.71–1.29 Ghanaian 1st generation 1.53 1.30–1.81 1.38 1.14–1.68 1.28 1.05–1.56 2nd generation 1.81 1.09–3.01 1.92 1.12–3.27 1.64 0.94–2.87 Turkish 1st generation 1.84 1.58–2.15 1.07 0.88–1.31 1.00 0.82–1.22 2nd generation 1.27 1.02–1.57 1.02 0.80–1.30 1.01 0.79–1.29 Moroccan 1st generation 1.81 1.55–2.11 1.22 1.00–1.49 1.15 0.94–1.41 2nd generation 1.14 0.92–1.41 0.93 0.73–1.19 0.89 0.69–1.14 Female sex 1.91 1.75–2.08 <.001 1.77 1.60–1.95 <.001 1.70 1.54–1.88 <.001 Age <.001 < 25 years Ref 25–34 years 1.04 0.87–1.24 35–44 years 1.28 1.09–1.50 45–54 years 1.34 1.15–1.56 55–64 years 1.42 1.21–1.66 ≥65 years 1.59 1.29–1.96 Educational level <.001 .005 .001 Unknown Ref Ref Ref No school/elementary school 1.41 0.95–2.09 1.55 0.93–2.58 1.55 0.87–2.75 Lower vocational/lower secondary school 1.05 0.71–1.55 1.50 0.90–2.50 1.47 0.83–2.60 Intermediate vocational/ intermediate secondary school 0.93 0.63–1.39 1.43 0.86–2.39 1.38 0.78–2.44 Higher vocational/university 0.66 0.44–0.99 1.18 0.71–1.99 1.12 0.63–2..00 Marital status <.001 Married/registered partnership Ref Cohabiting 0.66 0.56–0.77 Unmarried/never married 0.78 0.71–0.86 Divorced/separated 1.14 1.02–1.27 Widow/widower 1.26 0.98–1.64 Health status Self-reported medical conditions (previous 12 months) Table 4 Variables associated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to AHD (N = 15,007) (logistic regression analysis) Univariable Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior OR (95% CI) P-values aOR (95% CI) P-values aOR (95% CI) P-values Sociodemographics ated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to c regression analysis) .001 Schuts et al. Determinants of antibiotic use and number of prescriptions ≥half hour chest pain or dotter/bypass operation 1.78 1.60–1.98 <.001 1.28 1.13–1.45 <.001 1.24 1.09–1.40 .001 Severe heart condition 1.45 1.18–1.79 .001 Malignant disorder 1.93 1.52–2.47 <.001 1.33 1.02–1.74 .037 Severe or chronic fatigue 1.86 1.71–2.02 <.001 1.20 1.08–1.33 .001 1.16 1.04–1.29 .008 High blood pressure 1.36 1.25–1.49 <.001 Artery stenosis 1.46 1.25–1.70 <.001 0.80 0.67–0.96 .014 0.77 0.64–0.92 .004 Respiratory diseases 2.19 1.96–2.44 <.001 1.66 1.47–1.87 <.001 1.59 1.41–1.81 <.001 Serious/persistent intestinal disorders 1.87 1.64–2.12 <.001 1.24 1.07–1.43 .004 1.22 1.05–1.41 .009 Psoriasis 1.28 1.05–1.56 .015 (Chronic) eczema 1.30 1.15–1.47 <.001 Incontinence 2.08 1.85–2.35 <.001 1.32 1.15–1.52 <.001 1.32 1.15–1.52 <.001 Body Mass Index <.001 < 18.5 Ref 18.5–25 1.03 0.72–1.46 25–30 1.24 0.87–1.76 30–40 1.64 1.15–2.33 ≥40 1.97 1.31–2.97 Smoking .184 <.001 .003 Yes Ref Ref Ref No, never 0.99 0.90–1.09 0.78 0.69–0.87 0.82 0.72–0.92 No, but ever 0.90 0.80–1.02 0.91 0.79–1.04 0.93 0.81–1.07 Alcohol usage <.001 .017 .012 Never Ref Ref Ref Not in previous 12 months 0.88 0.77–1.02 0.96 0.82–1.12 0.95 0.81–1.12 Monthly or less 0.68 0.61–0.77 0.83 0.72–0.95 0.82 0.71–0.94 2–4 times per month 0.73 0.64–0.83 0.93 0.79–1.09 0.92 0.78–1.08 2–3 times per week 0.64 0.55–0.75 0.92 0.76–1.10 0.89 0.74–1.08 ≥4 times per week 0.48 0.39–0.58 0.70 0.55–0.88 0.68 0.54–0.86 Difficulty with Dutch language <.001 No Ref Yes 1.32 1.21–1.43 Not applicable 0.80 0.71–0.91 Perceived health <.001 <.001 .002 Excellent Ref Ref Ref Very good 1.09 0.86–1.37 1.05 0.83–1.34 1.03 0.80–1.31 Good 1.55 1.27–1.90 1.22 0.99–1.51 1.20 0.97–1.49 Mediocre 2.47 2.01–3.04 1.37 1.09–1.72 1.32 1.05–1.67 Bad 3.85 3.02–4.91 1.69 1.28–2.23 1.59 1.20–2.11 Antibiotic-related behavior Table 4 Variables associated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to AHD (N = 15,007) (logistic regression analysis) (Continued) Univariable Multivariable excluding Multivariable including ated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to c regression analysis) (Continued) Schuts et al. Determinants of antibiotic use and number of prescriptions Antimicrobial Resistance and Infection Control (2019) 8:200 Page 13 of 17 Page 13 of 17 Table 4 Variables associated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to AHD (N = 15,007) (logistic regression analysis) (Continued) Univariable Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior OR (95% CI) P-values aOR (95% CI) P-values aOR (95% CI) P-values No Ref Yes 0.77 0.71–0.84 Ever asked GP for antibiotics <.001 <.001 No, never Ref Ref Yes, regularly 4.72 3.59–6.21 3.07 2.28–4.14 Yes, occasionally 2.42 2.20–2.66 2.11 1.91–2.34 Did not finish treatment <.001 <.001 Always finished or no antibiotics Ref Ref Yes, regularly 2.70 2.02–3.62 1.80 1.29–2.50 Yes, occasionally 1.62 1.45–1.83 1.32 1.16–1.50 Abbreviations: OR Odds Ratio, aOR adjusted Odds Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner ated with having received ≥1 antibiotic prescription in the year prior to HELIUS visit in participants linked to c regression analysis) (Continued) ethnic groups received less antibiotics for viral infections than non-Hispanic white children. antibiotic prescriptions was second generation Ghanaian participants. Furthermore, we showed that a lower level of antibiotic knowledge was not associated with receiving antibiotics or average number of antibiotic prescriptions, and that ethnic differences in antibiotic use therefore cannot be explained by level of know- ledge on antibiotics. Lower odds for higher level of antibiotic use know- ledge were also seen among individuals who regularly or occasionally requested antibiotics from their GP or who regularly or occasionally did not finish treatment. These findings suggest that improving antibiotic knowledge might decrease the number of requests for antibiotics in primary care and improve appropriate use. g A previous study in Dutch primary care centres dem- onstrated higher use of antibiotics among first- generation migrants from Turkey, Morocco, Surinam or the Antilles compared to Dutch, after adjustment for age, sex, education, presence of chronic diseases, and smoking [3]. We found that the odds of having ≥1 anti- biotic prescription was higher in some ethnic groups in unadjusted analysis, but after adjusting for several vari- ables including medical conditions, the odds were sig- nificantly higher among Ghanaian and first-generation Moroccan participants only. In contrast, in our analyses on the number of antibiotic prescriptions as an outcome, only second-generation Ghanaian migrants were at higher risk of receiving a higher number of prescriptions compared to Dutch participants. Determinants of antibiotic use and number of prescriptions For all other ethnic groups, no evidence of a higher risk for more frequent prescriptions was found, while even a lower number was present for first-generation African Suriname and Turk- ish participants. To the best of our knowledge, no other studies have evaluated the variation in level of antibiotic knowledge and antibiotic use between ethnic groups and thus our findings need to be confirmed. Notably, our findings on antibiotic prescriptions and ethnicity are in line with a large retrospective cohort study performed in pediatric emergency departments in the United States [15]. This study also looked at the association between ethnicity and antibiotic prescribing, showing that other Our study has several strengths. First, the HELIUS study consists of a large number of participants from major ethnic groups living in the same city, with repre- sentation from all socioeconomic levels. Second, all out- comes and determinants were measured using the same methodology across all ethnic groups and HELIUS used translated questionnaires and had ethnically-matched in- terviewers and research assistants to provide assistance during data collection. These procedures enhance the comparability between ethnic groups. Another major strength of the current study is that HELIUS data could be linked to data from a health insurance register cover- ing the majority (77.7%) of the study population. g j y y p p Our study has also limitations. First, although HELIUS participants were recruited via an ethnicity-stratified random selection of the municipal registry of Amsterdam, the response rate for HELIUS study was 28% and there may be selection bias [8]. However, ana- lysis from a previous HELIUS study have shown that participants are not exceedingly different from non- respondents regarding sociodemographic variables [8]. Second, we did not take into account the use of antibi- otics purchased over the counter in the home country of participants [6, 16–18], and we might therefore have underestimated antibiotic use in non-Dutch ethnic Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 14 of 17 Schuts et al. Determinants of antibiotic use and number of prescriptions Antimicrobial Resistance and Infection Control (2019) 8:200 Page 14 of 17 Page 14 of 17 Table 5 Variables associated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated negative binomial regression analysis) Univariablea Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior IRR (95% CI) P-values IRR (95% CI) P-values IRR (95% CI) P-values Sociodemographics Ethnicity .004 .004 .001 Dutch Ref Ref Ref South-Asian Surinamese 1st generation 1.06 0.85–1.31 0.86 0.68–1.10 1.02 0.82–1.28 2nd generation 0.82 0.55–1.21 0.94 0.61–1.44 0.94 0.64–1.36 African Surinamese 1st generation 0.79 0.64–0.99 0.73 0.58–0.93 0.81 0.65–1.00 2nd generation 0.83 0.57–1.22 0.90 0.59–1.36 0.93 0.65–1.33 Ghanaian 1st generation 0.75 0.60–0.94 0.77 0.60–1.00 0.81 0.65–1.02 2nd generation 1.66 0.89–3.11 2.09 1.06–4.12 2.70 1.47–4.94 Turkish 1st generation 0.85 0.69–1.04 0.74 0.59–0.92 0.79 0.64–0.97 2nd generation 1.14 0.87–1.51 1.14 0.84–1.53 1.10 0.84–1.44 Moroccan 1st generation 0.99 0.81–1.21 0.89 0.70–1.11 0.89 0.72–1.10 2nd generation 0.84 0.63–1.13 0.92 0.67–1.27 1.02 0.76–1.37 Female sex 1.36 1.20–1.54 <.001 1.35 1.18–1.54 <.001 1.29 1.15–1.46 <.001 Age .023 < 25 years Ref 25–34 years 1.09 0.91–1.30 35–44 years 1.16 0.98–1.38 45–54 years 1.09 0.92–1.29 55–64 years 1.14 0.95–1.36 ≥65 years 1.44 1.16–1.80 Educational level .096 Unknown Ref No school/elementary school 1.74 0.86–3.52 Lower vocational/lower secondary school 1.74 0.86–3.52 Intermediate vocational/ intermediate secondary school 1.57 0.77–3.17 Higher vocational/university 1.45 0.71–2.96 Marital status .212 Married/registered partnership Ref Cohabiting 0.85 0.72–1.00 Unmarried/never married 0.99 0.89–1.10 Divorced/separated 1.01 0.90–1.13 Widow/widower 1.14 0.90–1.45 Health status Self-reported medical conditions (previous 12 months) Table 5 Variables associated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated negative binomial regression analysis) Univariablea Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior IRR (95% CI) P-values IRR (95% CI) P-values IRR (95% CI) P-values S i d hi ociated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated gression analysis) ted with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated i l i ) Schuts et al. Determinants of antibiotic use and number of prescriptions Antimicrobial Resistance and Infection Control (2019) 8:200 Page 15 of 17 Page 15 of 17 Table 5 Variables associated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated negative binomial regression analysis) (Continued) Univariablea Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior IRR (95% CI) P-values IRR (95% CI) P-values IRR (95% CI) P-values CVA/one-sided loss of bodily function ≤1 day 1.10 0.95–1.29 .208 MI incl. ≥half hour chest pain or dotter/bypass operation 1.35 1.19–1.54 <.001 1.22 1.06–1.41 .005 Severe heart condition 1.26 1.04–1.52 .019 Malignant disorder 1.91 1.47–2.48 <.001 1.60 1.21–2.12 .001 1.60 1.28–2.00 Severe or chronic fatigue 1.29 1.15–1.44 <.001 High blood pressure 1.14 1.04–1.25 .006 Artery stenosis 1.30 1.08–1.57 .005 Respiratory diseases 1.50 1.32–1.71 <.001 1.34 1.16–1.54 <.001 1.29 1.13–1.47 <.001 Serious/persistent intestinal disorders 1.24 1.07–1.44 .004 Psoriasis 1.08 0.90–1.31 .406 (Chronic) eczema 1.21 1.07–1.37 .002 1.14 1.00–1.29 .042 Incontinence 1.31 1.13–1.50 <.001 Body Mass Index .736 < 18.5 Ref 18.5–25 0.98 0.70–1.37 25–30 0.98 0.70–1.37 30–40 1.04 0.74–1.46 ≥40 1.06 0.71–1.57 Smoking .099 Yes Ref No, never 1.13 1.00–1.29 No, but ever 1.02 0.87–1.21 Alcohol usage .075 Never Ref Not in previous 12 months 0.93 0.77–1.13 Monthly or less 0.94 0.80–1.11 2–4 times per month 1.01 0.85–1.20 2–3 times per week 0.73 0.58–0.91 ≥4 times per week 0.81 0.61–1.08 Difficulty with Dutch language .320 No Ref Yes 0.94 0.86–1.04 Not applicable 1.05 0.88–1.25 Perceived health <.001 .001 <.001 Excellent Ref Ref Ref Very good 0.98 0.68–1.42 1.03 0.71–1.49 0.99 0.70–1.39 Good 1.29 0.94–1.77 1.19 0.85–1.65 1.17 0.86–1.59 Mediocre 1.73 1.25–2.39 1.40 1.00–1.97 1.44 1.05–1.97 Bad 2.30 1.62–3.26 1.71 1.17–2.49 1.72 1.22–2.43 Antibiotic-related behavior ociated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated gression analysis) (Continued) .054 Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Page 16 of 17 Schuts et al. Abbreviations AHD: Achmea Health Database; aIRR: adjusted Incidence Risk Ratio; aOR: adjusted Odds Ratio; BMI: Body Mass Index; CVA: Cerebrovascular Accident; GP: General Practitioner; HELIUS: Healthy life in an Urban Setting; IQR: Interquartile Range; IRR: Incidence Risk Ratio; MI: Myocardial Infarction; OR: Odds Ratio; WHO: World Health Organization Determinants of antibiotic use and number of prescriptions Moreover, since this was a cross-sectional study, we were unable to model antibiotic knowledge with future antibiotic prescriptions. Further research should examine the association of anti- biotic knowledge with future antibiotic prescriptions. Fi- nally, we are unable to determine if individuals were more demanding towards their GP or if their GPs were more lenient in prescribing antibiotics during illness [4, 5]. Neither completing antibiotic therapy, assessed by pill count, nor duration of antibiotic use could be taken into account as these data were not il bl Determinants of antibiotic use and number of prescriptions Antimicrobial Resistance and Infection Control (2019) 8:200 Page 16 of 17 Page 16 of 17 Table 5 Variables associated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated negative binomial regression analysis) (Continued) Univariablea Multivariable excluding variables on antibiotic- related behavior Multivariable including variables on antibiotic- related behavior IRR (95% CI) P-values IRR (95% CI) P-values IRR (95% CI) P-values No Ref Yes 0.92 0.85–1.00 Ever asked GP for antibiotics <.001 <.001 No, never Ref Ref Yes, regularly 2.96 2.34–3.73 1.87 1.45–2.41 Yes, occasionally 1.75 1.59–1.92 1.15 1.06–1.30 Did not finish treatment <.001 Always finished or no antibiotics Ref Yes, regularly 1.50 1.12–2.00 Yes, occasionally 1.32 1.18–1.48 Abbreviations: IRR Incidence Risk Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner aAccounts for zero-inflated distribution ociated with number of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated gression analysis) (Continued) umber of antibiotic prescriptions in participants linked to ADH (N = 15,007) (zero-inflated s: IRR Incidence Risk Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner i fl d di ib i Abbreviations: IRR Incidence Risk Ratio, CI Confidence Interval, CVA Cerebro Vascular Accident, MI Myocardial infarction, GP General Practitioner aAccounts for zero-inflated distribution groups. As a recent HELIUS study found that Dutch people of Turkish or Moroccan origin were more likely to use healthcare in the Netherlands as well as their country of origin [19], underestimation of antibiotic use in non-Dutch ethnic groups seems unlikely. Third, since several characteristics, such as education level and med- ical conditions, of HELIUS participants insured at Ach- mea differed from those insured elsewhere, selection bias could have been introduced in analysis on antibiotic use. This difference could be due to the fact that the City of Amsterdam provided health insurance discounts with Achmea for low-income individuals. These differ- ences were corrected for during multivariable analyses to the most possible extent. Fourth, the variable ‘ever asked GP for antibiotics’ does not discriminate between appropriate or inappropriate requests for antibiotics and misclassification might have occurred. However, this variable gives some information on participants’ atti- tudes towards antibiotic use. Furthermore, due to pri- vacy restrictions, we were unable to include indication for antibiotic therapy and duration of antibiotic use as additional indices for antibiotic use (apart from the number of antibiotics prescribed). Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13756-019-0636-x. pp y Supplementary information accompan 1186/s13756-019-0636-x. Additional file 1. Supplementary Methods. Additional file 2: Table S1. Characteristics of participants not linked versus linked to the Achmea Health Database. Additional file 1. Supplementary Methods. Additional file 2: Table S1. Characteristics of participants not linked versus linked to the Achmea Health Database. Conclusions To our knowledge, this study is the first to examine eth- nic disparities in level of antibiotic knowledge and use in a large population-based sample among adults with dif- ferent ethnic backgrounds. Health policy makers and healthcare professionals are increasingly developing in- terventions to improve the quality of antibiotic use, which is needed to help contain antimicrobial resistance. Targeted campaigns can be considered, for instance, during the annual European Antibiotic Awareness Day, since this event addresses improvement in the quality of antibiotic use to the general public [20]. Still, this study shows that a lower level of antibiotic knowledge is not necessarily linked to higher antibiotic usage, indicating that interventions aimed at improving knowledge alone might be insufficient to reduce antibiotic use. Neverthe- less, the underlying reasons for these findings need further evaluation. Author details 1D f 20. European Centre for Disease Prevention and Control. Antimicrobial consumption. In: ECDC, editor. Annual epidemiological report for 2016. Stockholm: ECDC; 2018. https://ecdc.europa.eu/en/publications-data/ antimicrobial-consumption-annual-epidemiological-report-2016. 1Department of Internal Medicine, Division of Infectious Diseases, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, 1105, AZ, Amsterdam, The Netherlands. 2Department of Infectious Diseases, Public Health Service Amsterdam, Nieuwe Achtergracht 100, 1018, WT, Amsterdam, The Netherlands. 3INSERM, Sorbonne Université, Institut Pierre Louis d’Épidémiologie et de Santé Publique, Paris, France. 4Department of Public Health, Amsterdam UMC, University of Amsterdam, Amsterdam Public Health Research Institute, Amsterdam, The Netherlands. 5Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam UMC, University of Amsterdam, Amsterdam Public Health Research Institute, Amsterdam, The Netherlands. Consent for publication Not applicable. 18. Hu J, Wang Z. In-home antibiotic storage among Australian Chinese migrants. Int J Infect Dis. 2014;26:103–6. 18. Hu J, Wang Z. In-home antibiotic storage among Australian Chinese migrants. Int J Infect Dis. 2014;26:103–6. 19. Sekercan A, Snijder MB, Peters RJG, Stronks K. Is healthcare consumption in the country of origin among Moroccan and Turkish migrants of older age (55+) associated with less use of care in the Netherlands? Tijdschr Gerontol Geriatr. 2018;49(6):253–62. 19. Sekercan A, Snijder MB, Peters RJG, Stronks K. Is healthcare consumption in the country of origin among Moroccan and Turkish migrants of older age (55+) associated with less use of care in the Netherlands? Tijdschr Gerontol Geriatr. 2018;49(6):253–62. Funding h The HELIUS study is conducted by the Academic Medical Center Amsterdam and the Public Health Service of Amsterdam. Both organizations provided core support for HELIUS. The HELIUS study is also funded by the Dutch Heart Foundation (2010 T084), the Netherlands Organization for Health Research and Development (ZonMw) (200500003), the European Union (FP-7) (278901), and the European Fund for the Integration of non-EU immigrants (EIF) (2013EIF013). The authors were independent from funders and had full access to all data. The decision to submit for publication was uninfluenced by the funders. The authors of this study take full responsibility for the integ- rity of the data and the accuracy of the data analysis. 10. StatLine. Population key figures 2019. https://opendata.cbs.nl/statline/#/CBS/ nl/dataset/37296ned/table?ts=156888214600. 11. StatLine. Population data; age, migration background, gender and region. 2019. https://opendata.cbs.nl/statline/#/CBS/nl/dataset/37713/table?ts=156 8882255075. 12. Stronks K, Kulu-Glasgow I, Agyemang C. The utility of 'country of birth' for the classification of ethnic groups in health research: the Dutch experience. Ethn Health. 2009;14(3):255–69. 13. Data Protection Authority. Ministry of Health, Welfare and Sport. https:// autoriteitpersoonsgegevens.nl/en. 14. Special Eurobarometer 338: Antimicrobial Resistance (2010). http://ec. europa.eu/public_opinion/archives/eb_special_339_320_en.htm#338. Acknowledgements The authors would like to acknowledge the HELIUS participants for their contribution; the HELIUS team for data collection and management. Page 17 of 17 Page 17 of 17 Page 17 of 17 Schuts et al. Antimicrobial Resistance and Infection Control (2019) 8:200 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 31 July 2019 Accepted: 24 October 2019 Received: 31 July 2019 Accepted: 24 October 2019 Authors’ contributions 7. Deschepper R, Grigoryan L, Lundborg CS, Hofstede G, Cohen J, Kelen GV, et al. Are cultural dimensions relevant for explaining cross-national differences in antibiotic use in Europe? BMC Health Serv Res. 2008;8:123. ES, MS, SG, MP and JP were involved in the conception and design of the study. ES, EvD and MS were involved in the acquisition of data. EvD and AB analyzed the data. ES and EvD wrote the manuscript. ES, EvD, AB, MS, SG, MP and JP were major contributors in revising the manuscript. All authors read and approved the final manuscript. 8. Snijder MB, Galenkamp H, Prins M, Derks EM, Peters RJG, Zwinderman AH, et al. Cohort profile: the Healthy life in an urban setting (HELIUS) study in Amsterdam, The Netherlands. BMJ Open. 2017;7(12):e017873. 9. Stronks K, Snijder MB, Peters RJ, Prins M, Schene AH, Zwinderman AH. Unravelling the impact of ethnicity on health in Europe: the HELIUS study. BMC Public Health. 2013;13:402. Availability of data and materials h d d h d The datasets used in the current study are available from the corresponding author on request, contingent on approval from the HELIUS scientific committee. The datasets used in the current study are available from the corresponding author on request, contingent on approval from the HELIUS scientific committee. 15. Goyal MK, Johnson TJ, Chamberlain JM, Casper TC, Simmons T, Alessandrini EA, et al. Racial and Ethnic Differences in Antibiotic Use for Viral Illness in Emergency Departments. Pediatrics. 2017;140(2):e20170203 (4). 16. Lindenmeyer A, Redwood S, Griffith L, Ahmed S, Phillimore J. Recent migrants’ perspectives on antibiotic use and prescribing in primary care: a qualitative study. Br J Gen Pract. 2016;66(652):e802–e9. 16. Lindenmeyer A, Redwood S, Griffith L, Ahmed S, Phillimore J. Recent migrants’ perspectives on antibiotic use and prescribing in primary care: a qualitative study. Br J Gen Pract. 2016;66(652):e802–e9. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate The HELIUS study is conducted in accordance with the Declaration of Helsinki and has been approved by the AMC Ethical Review Board. All participants provided written informed consent. 17. Hu J, Wang Z. Non-prescribed antibiotic use and general practitioner service utilisation among Chinese migrants in Australia. Aust J Prim Health. 2016; 22(5):434–9. 17. Hu J, Wang Z. Non-prescribed antibiotic use and general practitioner service utilisation among Chinese migrants in Australia. Aust J Prim Health. 2016; 22(5):434–9. p. 257. 2. Nellums LB, Thompson H, Holmes A, Castro-Sanchez E, Otter JA, Norredam M, et al. Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis. Lancet Infect Dis. 2018;18(7):796–811. 3. Hogenhuis CC, Grigoryan L, Numans MM, Verheij TJ. Differences in antibiotic treatment and utilization of diagnostic tests in Dutch primary care between natives and non-western immigrants. Eur J Gen Pract. 2010;16(3):143–7. 4. Grigoryan L, Burgerhof JG, Degener JE, Deschepper R, Lundborg CS, Monnet DL, et al. Determinants of self-medication with antibiotics in Europe: the impact of beliefs, country wealth and the healthcare system. J Antimicrob Chemother. 2008;61(5):1172–9. 5. Grigoryan L, Burgerhof JG, Degener JE, Deschepper R, Lundborg CS, Monnet DL, et al. Attitudes, beliefs and knowledge concerning antibiotic use and self-medication: a comparative European study. Pharmacoepidemiol Drug Saf. 2007;16(11):1234–43. 6. Norris P, Ng LF, Kershaw V, Hanna F, Wong A, Talekar M, et al. Knowledge and reported use of antibiotics amongst immigrant ethnic groups in New Zealand. J Immigr Minor Health. 2010;12(1):107–12. References WHO A 1. WHO. Antimicrobial resistance: global report on surveillance 2014; 2014. p. 257. 2. Nellums LB, Thompson H, Holmes A, Castro-Sanchez E, Otter JA, Norredam M, et al. Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis. Lancet Infect Dis. 2018;18(7):796–811. y 3. Hogenhuis CC, Grigoryan L, Numans MM, Verheij TJ. Differences in antibiotic treatment and utilization of diagnostic tests in Dutch primary care between natives and non-western immigrants. Eur J Gen Pract. 2010;16(3):143–7. 4. Grigoryan L, Burgerhof JG, Degener JE, Deschepper R, Lundborg CS, Monnet DL, et al. Determinants of self-medication with antibiotics in Europe: the impact of beliefs, country wealth and the healthcare system. J Antimicrob Chemother. 2008;61(5):1172–9. 5. Grigoryan L, Burgerhof JG, Degener JE, Deschepper R, Lundborg CS, Monnet DL, et al. Attitudes, beliefs and knowledge concerning antibiotic use and self-medication: a comparative European study. Pharmacoepidemiol Drug Saf. 2007;16(11):1234–43. 6. Norris P, Ng LF, Kershaw V, Hanna F, Wong A, Talekar M, et al. Knowledge and reported use of antibiotics amongst immigrant ethnic groups in New Zealand. J Immigr Minor Health. 2010;12(1):107–12.
https://openalex.org/W4234104994	https://brill.com/downloadpdf/journals/ges/67/2/article-p217_3.pdf	French	null	Eléments pour une histoire de la communauté thérapeutique dans la psychiatrie occidentale de la seconde moitié du 20e siècle	Gesnerus	2,010	cc-by-sa	13,303	Gesnerus 67/2 (2010) 217–240 Gesnerus 67/2 (2010) 217–240 Summary Based on a critique of the traditional ruling of mental hospital, therapeutic community is an innovative model elaborated in Great Britain during World War II. According to this approach, all the relationships at work inside the institution have a big impact on the patients’ state. One of the favoured tools of the therapeutic community lies in regular meetings common to patients and staff, but also reserved to professionals. During these sessions small and big problems are intended to be discussed and resolved collectively. The constitution of this approach as a model and its diffusion in continental European psychiatry during the second half of the 20th century is described in this paper. Four stages are distinguished: the genesis, the constitution of a distinct approach and diffusion in Continental Europe, the radicalisation and criticism by the antipsychiatric movement, the institutionalisation and decline. Keywords: therapeutic community; 20th century psychiatry; Europe; psychosocial reform; antipsychiatry Keywords: therapeutic community; 20th century psychiatry; Europe psychosocial reform; antipsychiatry Eléments pour une histoire de la communauté thérapeutique dans la psychiatrie occidentale de la seconde moitié du 20e siècle Catherine Fussinger Catherine Fussinger, Institut universitaire d’histoire de la médecine et de la santé publique, 1, chemin des Falaises, CH-1005 Lausanne (Catherine.Fussinger@chuv.ch). Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 Résumé Basée sur une critique du système traditionnel de prise en charge des patient-e-s dans les hôpitaux psychiatriques, la communauté thérapeutique est un modèle novateur élaboré en Grande-Bretagne durant la Seconde Guerre mondiale, dans lequel l’ensemble des rapports interpersonnels à l’œuvre au sein de l’institution sont perçus comme ayant un fort impact Catherine Fussinger, Institut universitaire d’histoire de la médecine et de la santé publique, 1, chemin des Falaises, CH-1005 Lausanne (Catherine.Fussinger@chuv.ch). Gesnerus 67 (2010) 217 Gesnerus 67 (2010) 217 sur l’état des malades. Aussi, l’un des instruments privilégiés de la commu- nauté thérapeutique réside-t-il dans un système de réunions régulières entre soignant-e-s et soigné-e-s, mais aussi entre professionnel-le-s, au cours des- quels petits et grands problèmes doivent être débattus et résolus collective- ment. La constitution de cette approche comme un courant à part entière et sa diffusion au sein de la psychiatrie occidentale au cours de la seconde moitié du 20ème siècle est retracée dans cet article en opérant une distinction entre quatre étapes: genèse, constitution du courant et diffusion en Europe conti- nentale, appropriation et critique au sein du mouvement antipsychiatrique, institutionnalisation et déclin. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 1 Ce modèle reste par contre plus prégnant dans la prise en charge de personnes toxicomanes. Introduction Comme bon nombre de notions ayant eu cours au sein de la psychiatrie occidentale, la communauté thérapeutique s’avère difficile à appréhender. Si cela fut vrai dans les années 1950 à 1980, y compris dans le milieu psy- chiatrique, cela l’est davantage encore aujourd’hui. En effet, bien que ce modèle soit encore appliqué ici et là, il a cessé d’être investi comme une véritable alternative dans les institutions prenant en charge des personnes souffrant de troubles psychiques1. Pourtant, après la première utilisation du terme en 1946, la communauté thérapeutique est rapidement devenue un modèle dont la carrière a duré quarante ans au moins.De plus,bien que son essor ait été au départ très étroi- tement lié à la psychiatrie britannique, puis nord-américaine, des commu- nautés thérapeutiques furent créées dans nombre de pays occidentaux à par- tir du milieu des années 1960. La visibilité de ce courant demeura toutefois relative. En effet, si la mise en œuvre de cette approche au sein d’un hôpital psychiatrique exigeait un intense travail de négociations, ces discussions se déroulèrent essentiellement au niveau local. Elles n’impliquèrent donc pas de mobilisation d’ampleur nationale au sein de la profession psychiatrique, comme ce fut le cas pour d’autres approches novatrices en matière de poli- tique de santé mentale. Cette inscription locale ainsi que la durée de vie sou- vent éphémère des expériences engagées expliquent sans doute pourquoi la communauté thérapeutique constitue un épisode peu connu de l’histoire de la psychiatrie dans la seconde moitié du 20ème siècle – période qui,rappelons- le, demeure encore actuellement la moins étudiée en regard des 150 années précédentes. 1 Ce modèle reste par contre plus prégnant dans la prise en charge de personnes toxicomanes. 218 Gesnerus 67 (2010) L’historien-ne amené-e à constater le rôle joué par cette approche dans telle ou telle institution psychiatrique risque dès lors d’être confronté-e à plusieurs difficultés. Comme nous le verrons plus loin, la communauté thé- rapeutique est un «objet mou» reposant sur des définitions labiles. Mais surtout, dans la plupart des cas, sa mise en œuvre au niveau local ne s’est pas accompagnée de production de documents et le recours à l’histoire orale ne suffit pas toujours à pallier ce manque de sources écrites. De plus, la littéra- ture secondaire ne permet que partiellement de surmonter ces difficultés. Introduction L’historien-ne amené-e à constater le rôle joué par cette approche dans telle ou telle institution psychiatrique risque dès lors d’être confronté-e à plusieurs difficultés. Comme nous le verrons plus loin, la communauté thé- rapeutique est un «objet mou» reposant sur des définitions labiles. Mais surtout, dans la plupart des cas, sa mise en œuvre au niveau local ne s’est pas accompagnée de production de documents et le recours à l’histoire orale ne suffit pas toujours à pallier ce manque de sources écrites. De plus, la littéra- ture secondaire ne permet que partiellement de surmonter ces difficultés. Certes, la plupart des ouvrages traitant de l’évolution de la psychiatrie au 20ème siècle en Grande-Bretagne ou aux USA mentionnent la communauté thérapeutique, mais ils la présentent en quelques paragraphes seulement2. Quant aux travaux consacrés à ce sujet, ils portent essentiellement sur les prémisses de ce courant durant la guerre3, ainsi que sur ses promoteurs les plus connus4; quelques études s’emploient également à analyser des expé- riences locales5. Enfin, la communauté thérapeutique a suscité très tôt l’intérêt des sociologues et des ethnologues qui l’ont pour la plupart abordée à partir de la thèse du contrôle social6. Effectuées entre les années 1950 et 1980, ces recherches, d’un apport incontestable, sont toutefois susceptibles d’être classées aussi bien parmi les sources que dans la littérature secondaire7. Force est donc de constater l’absence de travaux historiques embrassant la carrière de la communauté thérapeutique dans son ensemble8. En retraçant les grandes étapes de son évolution,le présent article entend offrir des jalons à partir desquels des expériences singulières devraient être plus faciles à situer. 5 Sur le rôle de la communauté thérapeutique au sein d’un hôpital de jour londonien: Spandler 2006. Dans sa thèse sur le mouvement antipsychiatrique aux Pays–Bas, Gemma Blok examine le fonctionnement d’une communauté thérapeutique établie dans un hôpital psychiatrique néerlandais: Blok 2004. 5 Sur le rôle de la communauté thérapeutique au sein d’un hôpital de jour londonien: Spandler 2006. Dans sa thèse sur le mouvement antipsychiatrique aux Pays–Bas, Gemma Blok examine le fonctionnement d’une communauté thérapeutique établie dans un hôpital psychiatrique néerlandais: Blok 2004. 6 Volontiers qualifiée de foucaldienne aujourd’hui, cette perspective a de fait diverses sources d’inspiration. 3 Rose 1989; Jones 2004;Thalassis 2007; Harrison 2000. 2 Jones 1972; Busfield 1986; Grob 1994. ; ; 4 Millard 1996; Harrison 2000. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 5 Sur le rôle de la communauté thérapeutique au sein d’un hôpital de jour londonien: Spandler 2006. Dans sa thèse sur le mouvement antipsychiatrique aux Pays–Bas, Gemma Blok examine le fonctionnement d’une communauté thérapeutique établie dans un hôpital psychiatrique néerlandais: Blok 2004. Introduction Pour le sociologue Michael Bloor (1986), dans le cas de la communauté théra- peutique, elle peut soit être appliquée au niveau macro (les finalités réadaptatives de la communauté thérapeutique se trouvent alors soulignées),soit au niveau micro (l’examen porte alors les rapports de pouvoir se jouant au quotidien).Victor Sharp (1975) aurait été le premier à étudier la communauté thérapeutique sous l’angle du contrôle social. p q g 7 Suite à une première recherche menée dans la deuxième moitié des années 1950 (Rapoport 1960, 1974) destinée à aider les psychiatres à mieux théoriser leur pratique, plusieurs autres études furent réalisées au cours des années 1970 et 80 dans une perspective qui se voulait plus critique (Sharp 1975; Bloor et al. 1982, 1986, 1988; Wiley 1986; McKeganey/Bloor 1987; Manning 1976, 1989). g ) 8 Les sociologues Bloor, McKeganey, et Fonkert (1988), ainsi que Manning (1976, 1989) propo- sent de brefs historiques.Pour le point de vue de promoteurs de la communauté thérapeutique: Clark 1977; Campling/Haigh 1999. g ) 8 Les sociologues Bloor, McKeganey, et Fonkert (1988), ainsi que Manning (1976, 1989) propo- sent de brefs historiques.Pour le point de vue de promoteurs de la communauté thérapeutique: Clark 1977; Campling/Haigh 1999. Gesnerus 67 (2010) 219 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 L’histoire de la communauté thérapeutique peut être divisée en quatre phases.Le premier moment,quelque peu mythique,est celui des expériences fondatrices au cours de la Seconde Guerre mondiale. La seconde étape, de 1945 au milieu des années 1960, voit la constitution à proprement parler de ce courant à travers la multiplication des expériences au sein des pays anglo- saxons ainsi que leur théorisation dans diverses publications. La troisième phase,qui court du milieu des années 1960 à la fin des années 1970,comprend deux mouvements distincts. On assiste d’une part à une diffusion du modèle de la communauté thérapeutique dans plusieurs pays occidentaux. On observe d’autre part une appropriation de cette approche par le mouvement antipsychiatrique, dont il résulta une radicalisation de certaines pratiques mais aussi une remise en cause. A partir des années 1980 enfin, ce courant donne des signes d’institutionnalisation, les efforts déployés en ce sens ne semblent toutefois pas être parvenus à enrayer son déclin. 9 Bloch 1962, 234sq.; Clark 1964, 42sq.; Clark 1977, 553; Bleandonu 1970, 9sq.; Main 1983, 217. 10 Clark (1977, 54) affirme par exemple que «l’analyse sociale» constitue l’outil principal de la communauté thérapeutique. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 La communauté thérapeutique: un premier portrait Avant de retracer la trajectoire de la communauté thérapeutique, il importe de commencer par en dresser un portrait plus précis. En concurrence avec des termes apparus à la même époque – tels que «psychiatrie sociale», «psychothérapie de groupe», mais aussi «psychiatrie administrative» –, la communauté thérapeutique fut d’emblée une notion élastique, ainsi que le relevèrent plusieurs acteurs sociaux9. Les différentes conceptions de cette approche ont toutefois en commun l’attention accordée à l’impact de «l’en- vironnement social» sur l’état des patient-e-s. En effet, l’idée forte qui sous- tend la communauté thérapeutique est que si les règles de vie et l’ensemble des relations interpersonnelles à l’œuvre au sein des institutions psychia- triques s’avèrent en certains cas pathogènes, elles ont aussi un pouvoir thé- rapeutique. L’emprise croissante des conceptions psychanalytiques dans les années 1950 explique en partie l’importance accordée à cette composante rela- tionnelle. Cela dit, tous les promoteurs de la communauté thérapeutique ne se définissent pas comme psychanalystes et la plupart d’entre eux ont refusé de présenter ce courant comme une forme de psychothérapie à proprement parler. De plus, ils insistèrent souvent davantage sur la dimen- sion «sociale» de leur approche10. Dans la mesure où ce terme renvoie 220 Gesnerus 67 (2010) surtout à la prise en compte des rapports interpersonnels et à la dynamique de groupe, le «social» tend toutefois à se confondre avec le «psychologique» dans ce contexte. L’élaboration de la communauté thérapeutique s’inscrit dans le cadre des efforts déployés dès les années 1950 pour rompre avec le fonctionnement traditionnel des hôpitaux psychiatriques, dénoncé comme profondément aliénant pour les malades. Formulée par une frange de psychiatres réforma- teurs11, cette critique fut très vite à l’origine d’expériences alternatives. Par- tie prenante de ce mouvement, la communauté thérapeutique a ainsi parfois pu englober presque l’intégralité des réformes entreprises pour «humaniser» les hôpitaux psychiatriques (extension et diversification des activités occu- pationnelles et de réhabilitation; introduction de la mixité; tentative de réta- blissement des liens avec l’extérieur par le biais de sorties en ville,de vacances et d’un maintien des contacts avec la famille; restitution des objets person- nels; diminution ou suppression des pratiques d’enfermement et de conten- tion, etc.). 11 Parmi les ouvrages ayant eu beaucoup de retentissement, on peut citer Stanton Alfred & Schwartz Morris, The Mental Hospital.A study of Institutional Participation in Psychiatric Illness and Treatment, 1954 et Barton Russel, Institutional Neurosis, 1959. 12 Initialement conçu à l’intention des patient-e-s psychiatriques, ce modèle a par la suite également été préconisé pour la prise en charge de personnes dépendantes (alcool, drogue), délinquantes ou emprisonnées; ces développements ne seront toutefois pas abordés dans cet article. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 La communauté thérapeutique: un premier portrait Si les théoriciens de la communauté thérapeutique situèrent bien leur travail dans le cadre de ces réformes, ils se distinguèrent cependant par l’attention particulière accordée à l’amélioration des relations et de la communication entre les malades et les soignant-e-s, mais aussi entre les différentes catégories de soignant-e-s. De manière plus restrictive, la notion de communauté thérapeutique a pu être utilisée pour désigner le fonctionnement d’un petit groupe de malades (généralement entre 20 et 30) et de soignant-e-s adoptant des règles de vie en commun qui se voulaient en elles-mêmes thérapeutiques. De telles com- munautés furent parfois créées au sein d’une division d’un hôpital psychia- trique dans le cadre de projets pilotes, comme elles purent s’installer dans une maison individuelle dans une zone d’habitation ordinaire et se doter d’un statut institutionnel autonome (fondation, association)12. Dans un cas comme dans l’autre, on retrouve cependant certaines carac- téristiques communes dans ce qui se voulait une transformation radicale de l’organisation hiérarchique des institutions psychiatriques. Des réunions journalières, ou au moins pluri-hebdomadaires, réunissant l’ensemble des malades et du personnel constituent le trait le plus saillant du fonctionne- ment des communautés thérapeutiques. Basées sur une libre circulation 12 Initialement conçu à l’intention des patient-e-s psychiatriques, ce modèle a par la suite également été préconisé pour la prise en charge de personnes dépendantes (alcool, drogue), délinquantes ou emprisonnées; ces développements ne seront toutefois pas abordés dans cet article. Gesnerus 67 (2010) 221 Gesnerus 67 (2010) 221 de la parole, qualifiée à l’époque de «démocratique», ces rencontres étaient censées permettre d’aborder tous les problèmes et incidents survenus au sein de l’institution, ainsi que de formuler des propositions afin d’améliorer l’organisation de la vie communautaire13. Ce nouvel agencement avait pour but explicite d’amener le personnel et les patient-e-s à s’investir activement dans l’entreprise thérapeutique. Cela impliquait notamment, pour le per- sonnel, de se déprendre d’une attitude décrite comme autoritaire, voire répressive,mais aussi d’accepter d’être régulièrement remis en cause lors des discussions collectives – aussi bien par ses collègues que par les patient-e-s – et d’être en mesure de reconnaître ses erreurs comme ses difficultés person- nelles. Considérés comme des sujets à part entière,les malades se virent quant à eux octroyer un droit à la parole. Plutôt que de les cantonner dans une position «d’objet passif de soins», on se mit à leur prêter des compétences thérapeutiques, tant pour eux-mêmes que pour leurs congénères. La communauté thérapeutique: un premier portrait L’octroi d’un tel statut s’accompagna toutefois de nouvelles exigences: on attendit en particulier d’eux qu’ils parviennent à s’affranchir de leur passivité comme de leur attitude oppositionnelle. Le paradoxe est que, pour parvenir à ce résultat,des comportements ouvertement oppositionnels furent tolérés,voire valorisés,ce qui impliquait un changement de posture considérable de la part de l’équipe soignante. Comme le soulignèrent deux psychiatres de l’époque: «La question la plus importante à se poser lorsqu’un malade est agité ou difficile est: Qu’est-ce que ce comportement signifie? et non: Comment pouvons-nous y mettre fin le plus aisément possible?»14 La permissivité qui résulta de ce repositionnement constitue une autre caractéristique impor- tante de la communauté thérapeutique. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 Genèse en temps de guerre Instituées rétrospectivement comme fondatrices, les premières expériences se déroulèrent en Grande-Bretagne entre 1941 et 1945, dans le contexte particulier de la Seconde Guerre mondiale15. Dans un climat où la question du moral et de la cohésion des troupes se voyait très investie,les services psy- 13 Ainsi que Clark (1977, 559) le souligne, l’étendue des domaines discutés collectivement a cependant pu varier considérablement d’une expérience à l’autre: si en certains cas l’équipe thérapeutique et les patient-e-s allèrent jusqu’à élaborer collectivement des décisions concer- nant les traitements, ces discussions communes furent parfois limitées à l’organisation des loisirs. 13 Ainsi que Clark (1977, 559) le souligne, l’étendue des domaines discutés collectivement a cependant pu varier considérablement d’une expérience à l’autre: si en certains cas l’équipe thérapeutique et les patient-e-s allèrent jusqu’à élaborer collectivement des décisions concer- nant les traitements, ces discussions communes furent parfois limitées à l’organisation des loisirs. 14 Le psychiatre belge Claude Bloch cite ici le psychiatre britannique Denis V. Martin: Bloch 1962, 248. 15 Cette première phase est celle qui a été le plus étudiée:Rose 1989;Harrison 2000;Jones 2004; Thalassis 2007. 222 222 Gesnerus 67 (2010) Gesnerus 67 (2010) chiatriques militaires britanniques furent le théâtre de pratiques novatrices qui impliquèrent des patients souffrant de névroses de guerre. Dès 1941, le psychiatre Maxwell Jones (1907–1990)16 – habituellement salué comme le père de la communauté thérapeutique – introduisit une série de modifications dans sa manière de traiter les patients hospitalisés à Mill Hill, une dépendance du Maudsley Hospital, dans la banlieue de Londres17. Responsable d’une unité psychosomatique réunissant une centaine de mili- taires souffrant de troubles fonctionnels cardiaques, Jones pensa que le meilleur moyen de les amener à admettre qu’ils souffraient de troubles psy- chosomatiques était de les informer des mécanismes à l’œuvre derrière leurs symptômes. Le nombre des patients ne lui permettant pas de s’entretenir individuellement avec chacun, il organisa des conférences à l’intention de tous. Il constata cependant rapidement que celles-ci n’étaient guère pro- fitables.Aussi décida-t-il de travailler en petits groupes, à raison de trois ren- contres par semaine durant un mois18. Ayant observé que certains aspects de la vie au sein des divisions étaient abordés par les patients lors de ces réunions, Jones s’intéressa aux «phénomènes sociologiques»19 à l’origine des changements d’atmosphère au sein des services. 16 Maxwell Jones est resté durant toute sa carrière un fervent promoteur de la communauté thérapeutique, voir Millard 1996 et Clark 2005. 17 Jones 1953, 1–15. 18 On trouve plusieurs transcriptions de ces séances de discussion dans Jones 1953, 4–12. 19 Jones 1953, 13. 20 Bion/Rickman 1943. 16 Maxwell Jones est resté durant toute sa carrière un fervent promoteur de la communauté thérapeutique, voir Millard 1996 et Clark 2005. p q 17 Jones 1953, 1–15. 19 Jones 1953, 13. 20 Bion/Rickman 1943. 7 Jo es 953, 5. 18 On trouve plusieurs transcriptions de ces séances de discussion dans Jones 1953, 4–12. 19 J 1953 13 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 Genèse en temps de guerre Ces différents constats amenèrent Jones et son équipe à entreprendre dès 1944 des jeux de rôles qui s’apparentaient au psychodrame de Jakob Moreno (1889–1974) – dont ils ignoraient cependant tout alors –, afin d’amener les patients à prendre conscience de leurs problèmes personnels. La seconde expérience se déroula en deux étapes et sous la houlette de plusieurs psychiatres, au sein d’un hôpital psychiatrique militaire doté de 300–400 lits,situé à Northfield près de Birmingham.Alors que Maxwell Jones put développer son projet durant presque toute la période de la guerre, la première expérience de Northfield,initiée en 1942,fut interrompue après six semaines par la direction de l’hôpital. En dépit de ce désaveu, les psychiatres et psychanalystes Wilfred Bion (1897–1979) et John Rickman (1891–1951) décidèrent de publier un article en commun, paru en 1943 dans The Lancet, afin de rendre compte du travail initié par chacun d’eux à Northfield20. On mesure la différence de leur démarche respective lorsqu’on sait que Bion était placé à la tête d’un training ward de 100 patients alors que Rickman entreprit une thérapie de groupe au sein d’une division qui ne comportait qu’une quinzaine de lits. Tous deux cependant entendaient trouver des Gesnerus 67 (2010) 223 Gesnerus 67 (2010) 223 Gesnerus 67 (2010) 223 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 moyens pour parvenir à établir un esprit de corps, conduisant chaque patient à contribuer de manière active au bon fonctionnement du groupe.C’est ainsi que Bion, par exemple, réduisit au minimum les exigences relatives au fonc- tionnement du service et y toléra un désordre certain, afin que la demande de règles de vie en commun émane des patients eux-mêmes plutôt que d’être imposée par les soignants. Bien que les expériences de Bion et de Rickman aient été interrompues prématurément, le travail de psychothérapie de groupe se poursuivit à Northfield avec l’arrivée en avril 1943 du psychanalyste Heinrich Foulkes (1898–1976). Peu avant la fin de la guerre, le psychiatre et psychanalyste Thomas Main (1911–1990) et Harold Bridger (1909–2005) – professeur de mathématiques avant le début du conflit – initièrent ce qu’on désigne habi- tuellement comme la seconde expérience de Northfield. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 21 Six articles paraissent dans le numéro de mai 1946 du Bulletin of the Menninger Clinic. 22 Main 1946, 66. 25 Rattaché jusqu’en 1959 au Belmont Hospital,un grand hôpital de la banlieue sud de Londres, le service initialement dirigé par Jones a reçu différentes appellations au fil des années: désigné dans un premier temps sous le terme de Industrial Neurosis Unit, il prit le nom de Social Rehabilitation Unit de 1954 à 1959; à partir de 1959, une fois ce service devenu indé- pendant, Henderson Hospital devint l’appellation définitive. Genèse en temps de guerre C’est à son propos que fut pour la première fois employé le terme de «communauté thérapeu- tique» par Main dans le cadre d’un numéro consacré, en 1946, à la psychia- trie britannique durant la guerre par une revue de psychiatrie américaine d’obédience psychanalytique21. Bien que moins véhémente que ce qu’elle sera au plus fort de l’antipsy- chiatrie, la critique du fonctionnement habituel des hôpitaux psychiatriques d’alors apparaît clairement dans l’introduction de l’article de Main: The concept of a hospital as a refuge too often means, however, that the patients are robbed of their status as responsible human beings.Too often they are called «good» or «bad» only according for their obedience, dependency and gratitude. The fine traditional mixture of charity and discipline they receive is a practised technique of removing their initiative as adult beings, and making them «patients». They are less trouble thus to the staff. [...] So, isolated anddominated,the patienttends to remain gripped by the hospital machine even in the games or prescribed occupations which occupy his time between treatments.Within such a setting, health and stability are too often bought at the excessive price of desocialisation. Sooner or later the patient,alone and unsupported,must face the difficult task of returning to the society in which he became unstable, and there regain social integration, and a daily sense of values and purpose. This task is no light for a desocialised man, however healthy he may have become.The design of a hospital as a social retreat also ignores positive therapeutic forces – the social support, and emotional opportunities which are granted in spontaneously struc- tured communities.22 L’impossibilité matérielle de pratiquer des psychothérapies approfondies au sein des institutions psychiatriques publiques ne constitue pas la seule raison pour Main de s’intéresser au potentiel thérapeutique de la communauté, il lui accorde en effet une pertinence intrinsèque: It is true that radical individual treatment can free the inner drives of the patient, and make him capable of full and stable social life, but it fails to give him an assured technique for full social participation – he can only learn this from the impact of society itself.Treatment of the neurotic patient, who suffers from a disturbance of social relationships, cannot therefore be 21 Six articles paraissent dans le numéro de mai 1946 du Bulletin of the Menninger Clinic. 22 Main 1946, 66. Genèse en temps de guerre 224 Gesnerus 67 (2010 224 224 Gesnerus 67 (2010) Gesnerus 67 (2010) regarded as satisfactory unless it is undertaken within a framework of social reality which can provide him with opportunities for attaining fuller social insight and for expressing and modifying his emotional drives according to the demands of real life.23 Dans le reste de son article, Main insiste beaucoup sur les changements de rôle que la mise en place d’une communauté thérapeutique implique tant de la part du psychiatre que du personnel infirmier.Ce thème,également présent d’emblée chez Maxwell Jones, a été abondamment repris par la suite. Notons que dans l’après-guerre ces deux psychiatres furent les seuls à œuvrer au développement de la communauté thérapeutique en tant que courant: Main comme psychanalyste,Jones comme promoteur de la psychiatrie sociale.Bion et Foulkes se tournèrent pour leur part vers le développement de la psycho- thérapie psychanalytique de groupe. Cela aurait sans doute également été le cas de Rickman s’il n’était pas décédé si rapidement. Quant à Bridger, il mit l’essentiel de ses compétences de psychanalyste au service du management. , 24 C’est du moins en ces termes que les patient-es de Jones sont présenté-e-s par un psychiatre suisse ayant fait un voyage d’étude en Grande-Bretagne:Schneider,1954,465;voir aussi Jones 1954. 23 Main 1946, 66. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 23 Main 1946, 66. 24 C’est du moins en ces termes que les patient-es de Jones sont présenté-e-s par un psychiatre suisse ayant fait un voyage d’étude en Grande-Bretagne:Schneider,1954,465;voir aussi Jones 1954. 25 Rattaché jusqu’en 1959 au Belmont Hospital,un grand hôpital de la banlieue sud de Londres, Constitution d’un courant Au cours de la seconde phase, de 1946 à 1965 environ, la communauté thérapeutique s’est progressivement imposée comme une approche en soi. Un nombre important de publications – des articles mais surtout des mono- graphies – relatant des expériences de plus longue durée fit connaître cette approche aux psychiatres en exercice à cette époque.Par ailleurs,après avoir été expressément présentée comme une approche adaptée aux malades névrotiques durant la guerre, elle fut étendue à d’autres catégories à partir des années 1950. De 1947 à 1959, Maxwell Jones s’employa à développer la commu- nauté thérapeutique en travaillant avec une population bien particulière de patient-e-s, qualifiés à l’époque de «psychopathes», de «déséquilibrés carac- tériels», d’«adultes asociaux» ou encore de «délinquants» – une catégorie de malades qui auraient été la «croix» des psychiatres, ces derniers ne sachant que faire pour les traiter24. Menée au sein d’une unité mixte de 100 lits (²/³ d’hommes pour un 1/³ de femmes) – le Belmont Hospital25 –, cette Gesnerus 67 (2010) 225 Gesnerus 67 (2010) 225 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 expérience a été présentée dans divers articles26 et dans deux ouvrages. Le premier, Social Psychiatry, est paru en 1952 en Grande-Bretagne sous la direction de Jones puis en 1953 sous un nouveau titre, The Therapeutic community. A New Treatment Method in Psychiatry, aux USA27. Le second fut publié en 1960 en Grande-Bretagne sous le titre de Community as doctor. New Perspectives on a Therapeutic Community28. Menée au sein du Belmont Hospital durant les années 1950 par une équipe placée sous la direction de Robert Norman Rapoport,un sociologue américain,cette étude visait à donner de meilleurs fondements théoriques à la communauté théra- peutique. Quelques années plus tard, le Dr Denis V. Martin29 montra pour sa part que la communauté thérapeutique était un mode de prise en charge suscep- tible d’être appliqué avec profit à l’ensemble des patient-e-s d’un hôpital psychiatrique ordinaire, soit aussi bien aux patient-e-s schizophrènes consi- déré-e-s comme chroniques qu’aux malades hospitalisé-e-s pour une crise aiguë, ou encore qu’aux névrosé-e-s. Le travail initié dès 1955 par Martin eut également un grand impact car,jusqu’alors,les communautés thérapeutiques avaient toujours été mises sur pied au sein d’unités comprenant une centaine de lits au maximum. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 ; p p ; ; 27 Si Maxwell Jones est présenté comme le premier auteur de ce travail, il fit figurer le nom de six collaborateurs comme coauteurs. 28 La version originale de cet ouvrage n’étant pas disponible, je me suis référée à la traduction française parue en 1974. 29 Malheureusement, je n’ai pas trouvé d’information sur la carrière du Dr Martin. 30 Ayant commencé à pratiquer la communauté thérapeutique dès 1958, le Dr Clark publia en 1964 Administrative Therapy.The role of the Doctor in the Therapeutic Community après avoir séjourné aux USA grâce à une bourse. On trouve plusieurs textes de David Clark sur l’histoire de la communauté thérapeutique et sur sa propre expérience à Fulbourn sur le site web des archives de The Planned EnvironmentTherapyTrust (http://www.pettarchiv.org.uk). 30 Ayant commencé à pratiquer la communauté thérapeutique dès 1958, le Dr Clark publia en 1964 Administrative Therapy.The role of the Doctor in the Therapeutic Community après avoir séjourné aux USA grâce à une bourse. On trouve plusieurs textes de David Clark sur l’histoire de la communauté thérapeutique et sur sa propre expérience à Fulbourn sur le site web des archives de The Planned EnvironmentTherapyTrust (http://www.pettarchiv.org.uk). 26 Jones 1954; Jones et Rapoport 1955; Jones 1956; Jones et al. 1956. p p 27 Si Maxwell Jones est présenté comme le premier auteur de ce travail, il fit figurer le nom de six collaborateurs comme coauteurs. 28 La version originale de cet ouvrage n’étant pas disponible, je me suis référée à la traduction française parue en 1974. 29 Malheureusement, je n’ai pas trouvé d’information sur la carrière du Dr Martin. 30 Ayant commencé à pratiquer la communauté thérapeutique dès 1958, le Dr Clark publia en 1964 Administrative Therapy.The role of the Doctor in the Therapeutic Community après 29 Malheureusement, je n’ai pas trouvé d’information sur la carrière du Dr Martin. ; p p ; ; 27 Si Maxwell Jones est présenté comme le premier auteur de ce travail, il fit figurer le nom de six collaborateurs comme coauteurs. 28 La version originale de cet ouvrage n’étant pas disponible, je me suis référée à la traduction française parue en 1974. 29 Malheureusement je n’ai pas trouvé d’information sur la carrière du Dr Martin Constitution d’un courant Or, après avoir dirigé à titre expérimental une commu- nauté thérapeutique de vingt-sept femmes schizophrènes, Martin parvint à étendre ce modèle à l’ensemble de Claybury, un hôpital de 2200 lits, situé à la périphérie de Londres. Décrivant les étapes de son travail et les difficultés rencontrées,Martin apportait la démonstration avec son livre – Adventure in Psychiatry. Social Change in a Mental Hospital (1962) – que la communauté thérapeutique n’était pas une approche réservée à des petites unités ac- cueillant des patient-e-s ayant un profil bien particulier comme cela avait été le cas jusqu’alors. Vers la fin des années 1950, un autre psychiatre, David H. Clark, entreprit d’introduire la communauté thérapeutique à Fulbourn, un hôpital psychia- trique de 820 lits, situé près de Cambridge30. Quant à Maxwell Jones, ce n’est qu’en 1962, après un séjour de trois ans aux USA, qu’il chercha à implan- 226 Gesnerus 67 (2010) ter son approche dans un hôpital psychiatrique «ordinaire» de 400 lits, au moment où il prit la direction de l’hôpital écossais de Dingleton, situé à 60 kilomètres d’Edimbourg31. Enfin, le Dr Thomas Main poursuivit quant à lui l’expérience ini- tiée durant la guerre en travaillant essentiellement avec des patient-e-s névrosé-e-s.Dès 1946,il prit en effet la direction d’une clinique psychiatrique privée, le Cassel Hospital, situé dans un comté au sud-est de Londres. Or, bien que cette clinique fût entrée en 1948 dans le National Health Service, elle avait pu conserver l’essentiel de ses caractéristiques antérieures. Elle demeura donc un établissement accueillant des névrosé-e-s, voire par- fois aussi la famille de ceux-ci. Mis à part le maintien de son orientation psychanalytique, les moyens matériels pour pratiquer des psychothérapies furent garantis par un taux d’encadrement des patient-e-s particulièrement élevé. En effet, au début des années 1960, près de quatorze psychiatres s’occupaient d’une soixantaine de patient-e-s32. De plus, non seulement la majorité des psychiatres avaient une formation analytique, mais c’était également le cas d’une bonne part du personnel infirmier33. En articulant traitements psychanalytiques individuels et communauté thé- rapeutique, Main eut surtout un rayonnement important au sein du mouvement psychanalytique, tandis que les travaux de Jones et de Martin montraient, pour leur part, que l’on pouvait pratiquer la communauté théra- peutique sans être psychanalyste34. Signalons enfin que c’est seulement à la fin de cette seconde phase que l’emploi du terme «communauté thérapeu- tique» s’imposa. 31 Bléandonu 1970, 38. Jones dirigea Dingleton jusqu’en 1969, date à laquelle il partit s’instal- ler définitivement aux USA, il était alors âgé de 62 ans. D’après son collègue David Clark, il tenta d’ouvrir des communautés thérapeutiques dans différents états, mais sans succès.A la fin de sa vie, il aurait développé un vif intérêt pour les questions spirituelles: Clark 2005. 32 Blé d 1970 56 32 Bléandonu 1970, 56. 36 http://pettrust.org.uk/index.php/Planned_Environment_Therapy_Trust 34 Jones a entrepris une psychanalyse et a également été supervisé dans les années 1950, il a toutefois renoncé à devenir membre de la société de psychanalyse britannique:Millard 1996; nous n’avons pas d’informations précises concernant le cursus de Martin, mais certains de ses propos nous conduisent à penser qu’il n’avait pas de formation analytique. 5 ili l d i l k l d , 33 Briggs 1959, 216. D’après Bloor/McKeganey/Fonkert 1988, le rôle thérapeutique des méde- cins et du personnel infirmier semble toutefois être demeuré clairement distinct (27). Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 35 Jones utilise les deux notions, Clark la seconde. 37 http://www.therapeuticcommunities.org 38 Manning 1989, 30sq. et 53. 39 Dans son ouvrage, Bléandonu (1970, 88–101) rend compte de la visite effectuée en 1967 par le Dr Roger Gentis dans trois communautés thérapeutiques néerlandaises. Voir aussi: Blok 2004. 40 Kennard 1999. 41 Ploeger 1980; Kennard 1999. 42 Ploeger 1980. 43 Legrand 1988, 15; Pietropoli Charmet 1997. 44 Pedrali 1997; Pietropoli Charmet 1997. Constitution d’un courant Auparavant, il s’était vu concurrencer par d’autres notions, comme celles de «psychiatrie sociale» ou de «psychiatrie administrative»35. Certains psychiatres américains parlèrent pour leur part de «thérapie par le milieu», ou encore d’«équipe thérapeutique». Au milieu des années 1960, la communauté thérapeutique avait suffisamment gagné en importance en Grande-Bretagne pour susciter la création d’une première association faîtière en 1966 – The Planned Environment Therapy Trust36 – puis d’une seconde en 31 Bléandonu 1970, 38. Jones dirigea Dingleton jusqu’en 1969, date à laquelle il partit s’instal- ler définitivement aux USA, il était alors âgé de 62 ans. D’après son collègue David Clark, il tenta d’ouvrir des communautés thérapeutiques dans différents états, mais sans succès.A la fin de sa vie, il aurait développé un vif intérêt pour les questions spirituelles: Clark 2005. 32 Blé d 1970 56 p ( ) 34 Jones a entrepris une psychanalyse et a également été supervisé dans les années 1950, il a toutefois renoncé à devenir membre de la société de psychanalyse britannique:Millard 1996; nous n’avons pas d’informations précises concernant le cursus de Martin, mais certains de ses propos nous conduisent à penser qu’il n’avait pas de formation analytique. 35 Jones utilise les deux notions, Clark la seconde. Gesnerus 67 (2010) 227 1972 – The Association ofTherapeutic Communities.37 En 1976,cette dernière comptait 30 membres collectifs représentant des institutions ayant adopté le modèle de la communauté thérapeutique, la moitié était établie en Grande- Bretagne et le reste à l’étranger et,10 ans plus tard,leur nombre avait doublé. D’après le sociologue Manning, 17 communautés thérapeutiques étaient en activité en Grande-Bretagne au milieu des années 7038. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 38 Manning 1989, 30sq. et 53. 37 http://www.therapeuticcommunities.org Diffusion en dehors de la Grande-Bretagne Sans parler des USA,où de nombreuses communautés thérapeutiques furent fondées, cette approche se diffusa dès les années 1960 en Europe continen- tale. Fragmentaires, les informations disponibles ne nous permettent toute- fois pas de préjuger de sa diffusion effective dans les différents pays. La communauté thérapeutique semble avoir été bien accueillie aux Pays- Bas. Une association nationale y fut notamment créée en 1973 à l’initiative de six hôpitaux39. De plus, bien acceptée par les instances dirigeantes de la psychiatrie, cette approche n’aurait pas été cantonnée dans une position marginale40. Les pays scandinaves lui auraient également réservé un bon accueil41. En Allemagne de l’Ouest par contre, où cinq communautés théra- peutiques au moins furent créées au cours des années 1960, la diffusion de ce modèle aurait été entravée – aux dires d’un de ses promoteurs – par le scepticisme conjoint des représentants de la psychiatrie universitaire et des médecins-directeurs des hôpitaux psychiatriques42. La communauté théra- peutique suscita également un enthousiasme certain en Italie. Ce modèle fut notamment adopté dès 1968 à Pérouse, deux communautés thérapeutiques furent créées à Milan dans la deuxième moitié des années 1960, ainsi qu’une à Rome et une autre à Venise43. Son développement semble toutefois avoir été en partie limité par la querelle entre des représentant-e-s de la commu- nauté thérapeutique accordant une grande importance aux conceptions psy- chanalytiques et les tenant-e-s de l’antipsychiatrie organisé-e-s dès 1973 au sein du mouvement Psichiatria Democratica, nous y reviendrons44. 228 228 Gesnerus 67 (2010) En France, la diffusion de la communauté thérapeutique fut sans doute inhibée par le statut qu’y occupait déjà la «psychothérapie institutionnelle»45. En effet, cette approche – également issue d’expériences menées durant la guerre – présente de nombreux points communs avec la communauté théra- peutique. Cette situation n’empêcha toutefois pas le courant britannique de faire l’objet d’une réception en France grâce à un travail éditorial conséquent initié par plusieurs psychiatres, le plus souvent déjà impliqués dans le déve- loppement de la psychothérapie institutionnelle. Le Dr Gérard Bléandonu fit ainsi plusieurs conférences et publications suite à deux voyages réalisés en Grande-Bretagne en 1966 et 1967 grâce à une bourse d’études46. Après avoir visité plusieurs communautés thérapeutiques néerlandaises, le Dr Roger Gentis présida également à la traduction d’une série de classiques, dont celle du livre du Dr Martin en 1969. 45 Chanoît 1995; von Bueltzingsloewen (à paraître); Coffin (à paraître); voir également les chapitres 2 et 3 de la thèse de Henckes 2007. 46 Bléandonu 1967, 1968, 1970, 1971. 47 Jones 1972 (é.o. anglaise 1968). 48 Bléandonu 1970, pp.102–105; Bléandonu s’appuie sur la thèse de médecine de Pierre Dessuant, La communauté thérapeutique.A propos d’une expérience concrète effectuée dans un hôpital psychiatrique, datant de 1969. 49 Woodbury, 1966, ch.VI. 46 Bléandonu 1967, 1968, 1970, 1971. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 47 Jones 1972 (é.o. anglaise 1968). 45 Chanoît 1995; von Bueltzingsloewen (à paraître); Coffin (à paraître); voir également les chapitres 2 et 3 de la thèse de Henckes 2007. Diffusion en dehors de la Grande-Bretagne Le Dr Paul Sivadon, expert pour l’Organisation Mondiale de la Santé, signa pour sa part une préface enthou- siaste pour l’édition française du troisième ouvrage de Maxwell Jones, parue en 197247. Une traduction de l’étude sociologique menée par Rapoport dans les années 1950 au Belmont Hospital fut également publiée en 1974 chez François Maspéro,un éditeur militant.Enfin,plusieurs articles de promoteurs de la communauté thérapeutique tels que Main, Jones ou encore Michael Woodbury furent également traduits dans L’Information psychiatrique, le journal du Syndicat des médecins des hôpitaux psychiatriques. En revanche, peu d’expériences concrètes furent entreprises. En 1970, Bléandonu n’avait connaissance que d’un seul projet, mené entre 1965 et 1968, au sein de l’Hôpital psychiatrique de Bassens (Savoie), alors dirigé par le Dr Henri Vermorel48. En Suisse – comme aux Pays-Bas, en Allemagne ou en Italie –, on dé- nombre au moins sept projets de communautés thérapeutiques initiés entre le début des années 1960 et le milieu des années 1980. Souvent en raison de conflits,la moitié d’entre eux durèrent deux ans à peine.Ce fut le cas du travail mené au sein du pavillon des Cèdres dans la clinique des Rives de Prangins (Vaud) entre 1962 et 1964 environ par le psychiatre américain MichaelWood- bury49, comme de l’expérience engagée dans l’une des divisions de l’Hôpital psychiatrique universitaire de Genève sous la houlette du Dr Bartold Bierens Gesnerus 67 (2010) 229 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 de Haan entre 1976 et 197750. On ignore par contre la durée de deux projets pilotes initiés au sein des institutions psychiatriques universitaires zuri- choises51. En 1963, le Dr Werner Stoll a mis sur pied une communauté thé- rapeutique modèle au sein d’une division pour femmes du Burghölzli52;ce fut également le cas du Dr Jürg Willi au sein de la division de psychothérapie stationnaire de la policlinique psychiatrique universitaire de Zürich en 196853. Les trois autres fonctionnèrent entre 10 et 25 ans. La communauté théra- peutique établie en 1965 au sein de la clinique privée du Schlössli (Zürich) par le Dr Edgar Heim semble avoir fonctionné jusqu’à sa nomination en 1979 à la chaire de psychothérapie de l’Université de Berne, dont il reprit égale- ment la direction de la policlinique psychiatrique54. 50 Steinauer 1982;Heimberg 2005;je me réfère également à des documents qui se trouvent dans les archives de l’hôpital psychiatrique de Malévoz, ainsi qu’à un échange de correspondance avec le Dr Bierens de Haan au printemps 2008. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 p p 51 Selon l’un des relecteurs anonymes de cet article, le Dr Ambros Uchtenhagen aurait égale- ment initié une communauté thérapeutique au début des années 1970 en collaboration avec l’association «Die Alternative» qui s’occupe de la prise en charge des personnes ayant des problèmes de drogues, sans avoir pour autant écrit sur cette expérience. 52 Germann 2007 233. 53 Kind 2000, 546. , 54 Heim 1978. Heim a également signé en 1976 la préface de la traduction allemande d’un des ouvrages de Maxwell Jones. 53 Kind 2000, 546. 54 Heim 1978. Heim a également signé en 1976 la préface de la traduction allemande d’un des ouvrages de Maxwell Jones. 55 Les renseignements concernant la communauté thérapeutique en Valais sont tirés d’une recherche en cours reposant sur des archives et des sources orales. 56 Aebi/Ciompi/Hansen 1993; Ciompi/Hoffmann/Broccard 2001; Ciompi 2005; http://www. soteria.ch 57 A ce jour, l’expérience de Kingsley Hall n’a pas fait l’objet d’une étude historique, aussi ce qu’on en sait repose essentiellement sur l’ouvrage relatant «le voyage à travers la folie» de Mary Barnes de son propre point de vue et de celui du psychiatre Joseph Berke qui l’a «accompagnée»: Barnes/Berke, 1971. Diffusion en dehors de la Grande-Bretagne De 1966 à 1990, soit durant près de vingt-cinq ans, la communauté thérapeutique fut égale- ment une référence centrale pour le Dr Jean Rey-Bellet et son adjoint le Dr Roberto Henking, tous deux placés à la tête de l’Hôpital psychiatrique de Malévoz (Valais)55. Au fil des années, la communauté thérapeutique y prit toutefois différentes formes: appliquée initialement dans l’ensemble des pavillons, elle fut surtout mise en œuvre dans celui réservé aux jeunes patient-e-s schizophrènes par la suite. Enfin, à Berne, la communauté théra- peutique de la Soteria accueille toujours des personnes en crise psychotique dans une petite maison d’un quartier résidentiel. Etablie en 1985 sous la forme d’une fondation autonome par le professeur de psychiatrie sociale d’alors, le Dr Luc Ciompi, ce projet semble s’être développé en bonne intel- ligence avec les institutions psychiatriques officielles du canton56. Mis à part la durée des expériences, on observe également des variations significatives dans le nombre de patient-e-s impliqué-e-s: près de 400 au Schlössli et à Malévoz, entre 10 et 20 dans les autres cas. De même, certaines de ces expériences se développèrent au sein d’institutions publiques (hôpi- taux psychiatriques du Valais, de Zurich et de Genève), alors que d’autres g 55 Les renseignements concernant la communauté thérapeutique en Valais sont tirés d’une recherche en cours reposant sur des archives et des sources orales. p 56 Aebi/Ciompi/Hansen 1993; Ciompi/Hoffmann/Broccard 2001; Ciompi 2005; http://www. soteria.ch 230 230 Gesnerus 67 (2010) Gesnerus 67 (2010) eurent pour cadre des cliniques ou des fondations privées (le Schössli, les Rives de Prangins et la Soteria). Enfin, si l’antipsychiatrie semble avoir constitué une référence positive à Genève et à la Soteria, il est probable que seule cette dernière ait opté pour l’usage des prénoms et l’abandon de la blouse blanche, une suppression des signes de hiérarchie entre personnel et soignant pourtant chère à Maxwell Jones. q 59 Clark,1977,555;pour une analyse des pratiques et des discours des promoteurs de la commu- nauté thérapeutique des années 1950 en regard de celles des antipsychiatres, voir Fussinger (à paraître). p g , 58 Bleandonu 1968, 749; Barnes/Berke 1971; Clark 1977, 555; Bloor/McKeganey/Fonkert 1988, 35sq. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 Radicalisation et remise en cause Au cours des années 1960, parallèlement à sa diffusion au sein des pays occidentaux, la communauté thérapeutique a également été investie par le mouvement antipsychiatrique et cela aussi bien en Italie qu’en Grande- Bretagne. En raison de la notoriété acquise par l’antipsychiatrie et de son autoreprésentation comme rupture radicale, peu de gens savent toutefois aujourd’hui que la communauté thérapeutique a constitué le cadre de travail initial aussi bien de Franco Basaglia (1924–1980), de Ronald Laing (1927– 1989) que de David Cooper (1931–1986). ) q p ( ) En Grande-Bretagne, la communauté thérapeutique de Kingsley Hall est considérée comme l’expérience fondatrice du mouvement antipsychia- trique57. Mené de 1965 à 1970, ce projet se trouve habituellement associé au nom de Ronald Laing, quand bien même d’autres psychiatres s’y impliquè- rent. Si plusieurs acteurs sociaux de l’époque ont présenté Kingsley Hall comme une forme particulière de communauté thérapeutique58 – dans laquelle le système des réunions régulières avait cédé la place à une organi- sation plus libertaire et où des comportements beaucoup plus transgressifs étaient tolérés59 –, sa notoriété provient essentiellement de son statut de projet alternatif radical. Le travail engagé entre 1962 et 1966 par le Dr David Cooper dans le cadre de la «Villa 21» – une petite division située dans un grand hôpital psychia- trique de 2000 lits – eut également un retentissement certain. Dans son ouvrage Psychiatry and Anti-Psychiatry paru en 1967, Cooper la présenta comme la première expérience antipsychiatrique menée en Grande-Bre- q 59 Clark,1977,555;pour une analyse des pratiques et des discours des promoteurs de la commu- nauté thérapeutique des années 1950 en regard de celles des antipsychiatres, voir Fussinger (à paraître). q 59 Clark,1977,555;pour une analyse des pratiques et des discours des promoteurs de la commu- nauté thérapeutique des années 1950 en regard de celles des antipsychiatres, voir Fussinger (à paraître). Gesnerus 67 (2010) 231 Gesnerus 67 (2010) 231 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 tagne. Pourtant, la description qu’il en donne nous montre que le fonction- nement de cette communauté thérapeutique accueillant une vingtaine de jeunes hommes, diagnostiqués schizophrènes, ressemblait à bien des égards à ce qui avait été décrit par un Maxwell Jones ou un Denis Martin. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 60 Cooper 1970 (é.o. anglaise 1967), 13. 61 Clark 1977, 554. 62 Fondée en 1964 par un petit groupe de psychiatres, parmi lesquels figuraient notamment Laing et Cooper, cette association a poursuivi ses activités après la fermeture de Kingsley Hall dont elle avait permis la mise sur pied; elle est toujours en activité aujourd’hui: http://www.philadelphia-association.org.uk 63 Cette association a été fondée en 1970 par des psychiatres qui s’étaient vu refuser l’accès à la Philadelphia Association, parmi lesquels se trouvait Joseph Berke (Burston 1996, 90–92), elle est également toujours en activité: http://www.arbourscentre.org.uk 64 Mosher 1991; Mosher 1999; Mosher/Hendrix/Fort, 2004; Bola/Mosher 2005. Plus globale- ment, sur la question du rapport des promoteurs de la communauté thérapeutique avec les traitements biologiques, notamment les psychotropes, voir Fussinger (à paraître). 65 Fussinger (à paraître). 62 Fondée en 1964 par un petit groupe de psychiatres, parmi lesquels figuraient notamment Laing et Cooper, cette association a poursuivi ses activités après la fermeture de Kingsley Hall dont elle avait permis la mise sur pied; elle est toujours en activité aujourd’hui http://www.philadelphia-association.org.uk 61 Clark 1977, 554. p p p g 63 Cette association a été fondée en 1970 par des psychiatres qui s’étaient vu refuser l’accès à la Philadelphia Association, parmi lesquels se trouvait Joseph Berke (Burston 1996, 90–92) elle est également toujours en activité: http://www.arbourscentre.org.uk 60 Cooper 1970 (é.o. anglaise 1967), 13. 64 Mosher 1991; Mosher 1999; Mosher/Hendrix/Fort, 2004; Bola/Mosher 2005. Plus globale- ment, sur la question du rapport des promoteurs de la communauté thérapeutique avec les traitements biologiques, notamment les psychotropes, voir Fussinger (à paraître). 65 Fussinger (à paraître). Radicalisation et remise en cause Si Cooper mentionne les emprunts faits à la communauté thérapeutique, il n’a de cesse de souligner le caractère unique et singulier de son expérience à la Villa 2160. Et force est de constater que cette lecture s’est rapidement imposée. L’analyse que fit Cooper de la fin de l’expérience de la Villa 21 eut égale- ment une incidence sur l’histoire de la communauté thérapeutique. Les conflits survenus avec la direction de l’hôpital psychiatrique ayant été à l’origine de cet échec, Cooper affirma que de véritables communautés thérapeutiques ne pouvaient se développer qu’en dehors de la psychiatrie officielle, sous forme de projets alternatifs autonomes, comme celui de Kingsley Hall. Si ce point de vue ne s’est pas imposé comme la nouvelle doxa parmi les représentant-e-s de ce courant, la question des avantages et des inconvénients respectifs des deux solutions fut par contre régulièrement débattue61. Après les années 1960, pas plus Laing que Cooper ne se distinguèrent par la création de nouvelles communautés thérapeutiques. Néanmoins, ce modèle continua à être promu par deux associations ayant les liens forts avec le mouvement antipsychiatrique: la Philadelphia Association62 et l’Arbors Association63. Parmi les héritages de Kingsley Hall, il faut encore mention- ner la communauté thérapeutique créée par le Dr Loren R. Mosher (1933– 2004) dans la Baie de San Fransisco (USA). En activité de 1971 à 1983 sous le nom de «Soteria House», ce projet se caractérise essentiellement par la volonté de traiter les crises psychotiques sans recourir aux neuroleptiques64. Du côté anglo-saxon, l’appropriation du modèle de la communauté thé- rapeutique par la mouvance antipsychiatrique s’est donc surtout traduite par une radicalisation de certaines pratiques ainsi que par une remise en cause systématique de tous les traitements somatiques65. En Italie, les rapports du g j p g 64 Mosher 1991; Mosher 1999; Mosher/Hendrix/Fort, 2004; Bola/Mosher 2005. Plus globale- ment, sur la question du rapport des promoteurs de la communauté thérapeutique avec les traitements biologiques, notamment les psychotropes, voir Fussinger (à paraître). 65 Fussinger (à paraître). 232 232 Gesnerus 67 (2010) mouvement antipsychiatrique avec la communauté thérapeutique prirent une tournure différente. Les débuts de l’antipsychiatrie dans ce pays sont habituellement assimilés au travail mené à partir de 1962 par Basaglia à Gorizia, un hôpital psychiatrique de 400 lits situé à la frontière italo-yougo- slave. Radicalisation et remise en cause Or, l’essentiel des efforts porta au départ sur la mise en œuvre des principes de la communauté thérapeutique, tels qu’ils s’étaient développés durant les années 1950 en Grande-Bretagne66. Ce n’est que dans un second temps que Basaglia et ses collaborateurs formulèrent ouvertement des ré- serves envers ce modèle, comme le montrent les propos du psychiatre Lucio Schittar parus dans L’Institution en négation – un ouvrage collectif édité en 1968 sous la direction de Basaglia en Italie. Le modèle lewinien de résolution des conflits sociaux a été appliqué à la communauté thérapeutique comme il l’avait été à l’administration et à l’industrie pour accroître leur efficacité. Selon lui «la participation» de chacun permet de réduire tous les conflits par la discussion; la ‹manipulation› de groupe permet d’émousser les contrastes les plus saillants par une attitude tolérante et compréhensive; la discussion de groupe oriente les efforts des participants,sous la conduite d’un leader éclairé,vers le ‹bon› objectif commun:qu’il s’agisse du déroulement ordonné du processus administratif, d’une production régulière, ou de la guérison-réhabilitation-intégration du malade mental. […] Le cycle semble clos. Ce qui était né comme une exigence de renouveau fondamental des institutions psychiatriques se révèle tout au plus, dans la pratique et en théorie, comme un nouveau type d’institution, plus moderne, plus efficace, mais où les rapports de pouvoir sont restés apparemment les mêmes. La ‹troisième révolution psychiatrique› ne serait donc qu’une adaptation tardive des moda- lités de contrôle social du comportement pathologique aux méthodes de production perfec- tionnées au cours des quarante dernières années par les sociologues et les techniciens de la communication de masse. Sous prétexte d’assainir les structures foncièrement déshumani- santes de l’asile d’aliénés, sociologues et psychologues de l’organisation semblent avoir trouvé le moyen d’appliquer au domaine de la psychiatrie institutionnelle les techniques (avant tout, des techniques de groupe) qui se sont révélées si efficaces dans la gestion de l’économie néocapitaliste, sans toucher cependant au pouvoir oppressif de la société67. Ces critiques ne furent toutefois pas à l’origine de la fin de la communauté thérapeutique à Gorizia. Cette expérience cessa suite aux démissions suc- cessives de Basaglia en 1968, puis du reste de son équipe en 1972. En effet, d’importants conflits avaient surgi avec les autorités locales, encore attisés par l’assassinat commis par un patient de l’hôpital psychiatrique en congé dans sa famille. 66 Basaglia 1966. Dans cet article, Basaglia se réfère explicitement à l’ouvrage de Denis V. Martin et aux travaux de Maxwell Jones. 67 La «troisième révolution psychiatrique» évoquée dans cette citation renvoie au statut revendiqué pour la «psychiatrie sociale» par les psychiatres réformateurs des années 1950, notamment par Maxwell Jones. Ne parlant pas l’italien, je me suis référée à la traduction française: Schittar 1970 (é.o. 1968) 148sq. Voir également dans cet ouvrage la contribution commune de Pirella/Slavich/Jervis/Basaglia, ainsi que celle de Slavich. 68 Rotelli 1982. 66 Basaglia 1966. Dans cet article, Basaglia se réfère explicitement à l’ouvrage de Denis V. Martin et aux travaux de Maxwell Jones. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 66 Basaglia 1966. Dans cet article, Basaglia se réfère explicitement à l’ouvrage de Denis V. Martin et aux travaux de Maxwell Jones. 67 La «troisième révolution psychiatrique» évoquée dans cette citation renvoie au statut revendiqué pour la «psychiatrie sociale» par les psychiatres réformateurs des années 1950, notamment par Maxwell Jones. Ne parlant pas l’italien, je me suis référée à la traduction française: Schittar 1970 (é o 1968) 148sq Voir également dans cet ouvrage la contribution 67 La «troisième révolution psychiatrique» évoquée dans cette citation renvoie au statut revendiqué pour la «psychiatrie sociale» par les psychiatres réformateurs des années 1950, notamment par Maxwell Jones. Ne parlant pas l’italien, je me suis référée à la traduction française: Schittar 1970 (é.o. 1968) 148sq. Voir également dans cet ouvrage la contribution commune de Pirella/Slavich/Jervis/Basaglia, ainsi que celle de Slavich. 68 Rotelli 1982 Radicalisation et remise en cause En dépit de ces réserves envers la communauté thérapeu- tique, Basaglia y recourut également lorsqu’il prit la direction de l’hôpital psychiatrique de Trieste en 197268. Assez rapidement toutefois, la fermeture Gesnerus 67 (2010) 233 Gesnerus 67 (2010) 233 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 des hôpitaux psychiatriques apparut comme une mesure plus pertinente que les efforts déployés pour leur transformation. Le modèle de la communauté thérapeutique se vit dès lors supplanté en Italie par la proposition de trans- férer l’ensemble de la prise en charge des patient-e-s psychiatriques dans des services ambulatoires établis dans la cité. Porté par une pluralité d’acteurs sociaux, ce projet se vit renforcé par la création en 1973 de l’Association Psichiatria Democratica.Ce mouvement joua en effet un rôle important dans le processus ayant conduit à l’adoption, en mai 1978, de la loi 180 qui pré- voyait la fermeture des hôpitaux psychiatriques en Italie. Concrètement, cette loi interdisait l’admission de nouveaux patients dans les hôpitaux psychiatriques. En lieu et place, chaque région devait créer un service extra- hospitalier de santé mentale à même de répondre à l’ensemble des besoins de la population (prévention, cure et réhabilitation). Lorsque des hospitali- sations s’avéraient indispensables,celles-ci devaient se faire dans des services de diagnostic et de traitements spécifiques établis au sein des hôpitaux généraux, dont la taille était limitée à 15 lits et dans lesquels la durée du séjour était limitée à sept jours, sauf prolongation explicite dûment justifiée. Si, au sein du courant Psichiatria Democratica, la communauté thérapeu- tique a parfois été considérée comme une transition nécessaire préparant une prise en charge entièrement extrahospitalière, elle s’est surtout trouvée critiquée comme une solution «technique» venant masquer l’exclusion sociale des malades. L’impact de l’antipsychiatrie sur la carrière de la com- munauté thérapeutique s’avère donc difficile à apprécier. Présenté comme une alternative progressiste au fonctionnement traditionnel des hôpitaux psychiatriques,ce courant a suscité un intérêt certain au sein des mouvements sociaux contestataires des années 1960–1970. En même temps, les critiques adressées à son endroit ont également amené les militant-e-s à s’en désinté- resser. Enfin, il n’est pas impossible que la popularité de la communauté thérapeutique au sein de la contre-culture ait joué en sa défaveur auprès des représentant-e-s de la psychiatrie officielle. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 69 Manning 1989. Institutionnalisation et déclin Durant les deux dernières décennies du 20ème siècle, les communautés théra- peutiques encore en activité entrèrent, selon le sociologue Manning, dans une phase de «professionnalisation et de routine»69.Pour le psychiatre David Clark toutefois, la communauté thérapeutique cessa d’être «à la mode» en 234 Gesnerus 67 (2010) 234 Gesnerus 67 (2010) Grande-Bretagne dès les années 1970.Aussi, la phase d’institutionnalisation tend ici à se confondre avec le mouvement de déclin70. Les difficultés ren- contrées furent de plusieurs ordres. Grande-Bretagne dès les années 1970.Aussi, la phase d’institutionnalisation tend ici à se confondre avec le mouvement de déclin70. Les difficultés ren- contrées furent de plusieurs ordres. Souvent tributaire du charisme d’un leader, la communauté thérapeuti- que – de par la nature même de son fonctionnement – semble avoir été un projet difficile à maintenir sur la durée. Basée sur une confrontation collec- tive continue, suscitant bien évidemment de nombreuses crises, l’expérience se révéla épuisante pour le personnel.A ces difficultés internes s’ajoutèrent des rapports conflictuels avec le milieu environnant et cela tant au sein des hôpitaux psychiatriques qu’en dehors de ceux-ci. Si dans les années 1960–1970, l’espoir d’un changement social radical très proche a réduit la portée de ces obstacles,le reflux de l’engagement militant à partir des années 1980 n’est pas resté sans incidences pour le courant de la communauté thé- rapeutique; il devint notamment rare de trouver des personnes prêtes à s’engager bénévolement dans un tel projet71. Par ailleurs, de l’avis des premiers intéressés, les réformes que connurent la plupart des systèmes de santé européens dans ces années-là se révélèrent très peu propices au maintien des communautés thérapeutiques72: avec le développement des prises en charge ambulatoires, les séjours de longue durée dans une institution thérapeutique assurant un hébergement étaient de moins en moins tolérés73. Enfin, si quelques commentateurs estiment qu’un certain déficit en matière de conceptualisation pesa défavorablement sur le développement de la communauté thérapeutique74, il fut surtout diffi- cile, dans ce cas comme dans bien d’autres,d’apporter la «preuve scientifique de l’efficacité de cette approche»75. Or, on le sait, de telles «preuves» furent exigées de plus en plus systématiquement au cours des deux dernières décennies du 20ème siècle. En réponse à ces difficultés, on observe durant cette période des efforts d’institutionnalisation. Ainsi, l’Association of Therapeutic Communities fonda en 1980 The International Journal for Therapeutic and Supportive Organizations. 70 Clark 1977. 71 Il semblerait notamment que tous les psychiatres qui travaillèrent à Kingsley Hall le firent de manière bénévole. En Italie aussi, des bénévoles s’engagèrent dans les structures psy- chiatriques alternatives créées pour la plupart dans le nord du pays. q p p 72 Manning 1989, 48; Kennard 1999, 243. 70 Clark 1977. 71 Il semblerait notamment que tous les psychiatres qui travaillèrent à Kingsley Hall le firent de manière bénévole. En Italie aussi, des bénévoles s’engagèrent dans les structures psy- chiatriques alternatives créées pour la plupart dans le nord du pays. 72 Manning 1989, 48; Kennard 1999, 243. 73 Notons toutefois que certains hôpitaux de jour adoptèrent le modèle de la communauté thé- rapeutique. 74 Manning 1989. 75 Clark 1977, 557sq. 70 Clark 1977. g 73 Notons toutefois que certains hôpitaux de jour adoptèrent le modèle de la communauté thé- rapeutique. 75 Clark 1977, 557sq. p q 74 Manning 1989. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 73 Notons toutefois que certains hôpitaux de jour adoptèrent le modèle de la communauté thé- rapeutique. 74 Manning 1989. 75 Clark 1977, 557sq. Institutionnalisation et déclin A l’instar de ce qui se pratiquait dans le domaine des psy- chothérapies, l’idée de formaliser un cursus de formation fut avancée, sans grand succès toutefois. Ainsi, un représentant de la communauté thérapeu- 70 Clark 1977. 71 Il semblerait notamment que tous les psychiatres qui travaillèrent à Kingsley Hall le firent de manière bénévole. En Italie aussi, des bénévoles s’engagèrent dans les structures psy- chiatriques alternatives créées pour la plupart dans le nord du pays. Gesnerus 67 (2010) 235 Gesnerus 67 (2010) 235 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 tique se demandait en 1999 quelle stratégie était envisageable pour permettre à ce courant de survivre. Si, en Grande-Bretagne, la communauté thérapeu- tique semble demeurer une référence vivante aujourd’hui encore, le travail de mémoire initié au sein de ce mouvement dénote une claire conscience de son déclin76.Ainsi,certaines associations britanniques commencent à dresser une liste des communautés thérapeutiques disparues tandis que d’autres lancent des appels à la constitution d’archives à même de documenter à l’avenir l’histoire de cette approche. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 76 Kennard 1999,244.Certaines associations britanniques ont par exemple produit une liste des communautés thérapeutiques disparues, une démarche qui se voit encadrée par des mesures visant à créer des archives. 77 On notera que la situation semble différente en ce qui concerne la prise en charge de la toxi- comanie, domaine dans lequel le modèle de la communauté semble une référence prégnante aujourd’hui encore. Ainsi l’European Federation of Therapeutic Communities, fondée en 1978 et toujours en activité aujourd’hui, se focalise sur ce type de prise en charge (http://www.eftc-europe.com). 78 Clark 1977, 563. 78 Clark 1977, 563. Bibliographie Aebi,Elisabeth/Luc Ciompi/Hansen Hartwig (Hrsg.),Soteria im Gespräch.Über eine alternative Schizophreniebehandlung (Bonn 1993) Basaglia, Franco, «La ‹communauté thérapeutique› base d’un service psychiatrique, réalités et perspectives», L’information psychiatrique 42 (1966) 413–428 p p f y ( ) Barnes, Mary/Joseph Berke, Mary Barnes. Un voyage à travers la folie (Paris 1973; é.o. anglaise 1971) ) Barton,Russel,La névrose institutionnelle (Paris 1969;é.o.anglaise:Institutional Neurosis 1959) Bi Wilf id R /J h Ri k “I i i h Th i d h k f h Bion, Wilfrid R./John Rickman, “Intra-group tensions in therapy. Their study as the task of the group”, The Lancet (1943) 678–681 Bleandonu, Gérard, «A la recherche de la communauté thérapeutique britannique (à propos d’un voyage d’étude)», L’information psychiatrique (1967) 1247–1266 Bleandonu, Gérard, «A la recherche de la communauté thérapeutique britannique (à propos d’un voyage d’étude)», L’information psychiatrique (1967) 1247–1266 – «La communauté thérapeutique (à propos d’une bourse d’étude du Conseil de l’Europe)», L’information psychiatrique 44 (1968) 739–751 Les communautés thérapeutiques (Paris 1970) Bleandonu, Gérard, «A la recherche de la communauté thérapeutique britannique (à propos d’un voyage d’étude)», L’information psychiatrique (1967) 1247–1266 – «La communauté thérapeutique (à propos d’une bourse d’étude du Conseil de l’Europe)», L’information psychiatrique 44 (1968) 739–751 – Les communautés thérapeutiques (Paris 1970) – «Les communautés thérapeutiques», Revue pratique de psychologie de la vie sociale et d’hygiène mentale 1 (1971) 73 88 (suivi d’un débat où sont reproduites notamment les y g ) f p y q ( ) – «La communauté thérapeutique (à propos d’une bourse d’étude du Conseil de l’Europe)» L’information psychiatrique 44 (1968) 739–751 – «Les communautés thérapeutiques», Revue pratique de psychologie de la vie sociale et d’hygiène mentale 1 (1971) 73–88 (suivi d’un débat où sont reproduites notamment les interventions des Drs Tosquelles, Oury 89–99) q y ) Bloch, Claude, «Aspects de l’assistance psychiatrique en Grande-Bretagne», L’information psy- chiatrique 38 (1962) 233–248, 355–363, 435–439 q ( ) Blok, Gemma, Baas in eigen Brein. «Antipsychiatrie» in Nederland, 1965–1985 (Amsterdam 2004) ) Bloor, Michael. J., “Social control in the therapeutic community: re-examination of a critical case”, Sociology of Health and Illness 8 (1986) 305–324 Bloor,Michael J./J.Dick Fonkert,“Reality construction,reality exploration and treatment in two therapeutic communities”, Sociology of Health and Illness 4 (1982) 125–140 Bloor,Michael/Neil McKeganey/Dick Fonkert,One foot in Eden:a sociological study of the range of therapeutic community practice (London 1988) Bola, R. John/Loren R. En guise de conclusion De l’avis même de certains de ses représentants,le modèle de la communauté thérapeutique semble avoir rencontré des difficultés à s’adapter aux trans- formations survenues au cours de deux dernières décennies du 20e siècle au sein du champ psychiatrique77. Doit-on pour autant conclure que cette approche s’est peu à peu éteinte sans laisser de traces? Lorsqu’en 1977,Clark établit le bilan des trente premières années d’existence de ce courant, il lui paraît pourtant évident que bon nombre des pratiques alors en vigueur dans les institutions psychiatriques britanniques lui sont redevables: A number of components of the therapeutic community method have become part of standard psychiatric practice. The majority staff, even in general hospitals, do not now wear uniform.A style of openness, first-naming and decision-making by consensus is widely used. Although ‹patient government› is not so widely used, ward meetings for the sharing of news and the planning of activities are very widespread. Group therapy, sociodrama and psy- chodrama, conducted by others than doctors and professional psychotherapists, is now quite common, and the number of professional staff experienced in group work is steadily rising.78 Un tel constat vaut-il pour l’ensemble des pays occidentaux? Les pratiques qui semblaient bien établies dans les années 1970 ont-elles perduré? Si la question reste ouverte en ce qui concerne l’abandon de la blouse blanche ou l’usage des prénoms, il est indéniable que la communauté thérapeutique a contribué à une réelle transformation de la culture interne aux institutions psychiatriques. En refusant le système hiérarchique pyramidal dominant dans les hôpitaux psychiatriques du milieu du 20ème siècle et en exigeant 236 236 Gesnerus 67 (2010) Gesnerus 67 (2010) une communication plus horizontale, ce courant a en effet fait le lit de pra- tiques parfaitement banales aujourd’hui telles que les réunions d’équipe ou les colloques réunissant soigné-e-s et soignant-e-s. Aussi, bien que la com- munauté thérapeutique soit une innovation plus difficile à cerner que d’autres, elle a toute sa place dans l’histoire de la psychiatrie de la seconde moitié du 20ème siècle et l’on ne peut que souhaiter que de nouvelles études historiques, basées notamment sur des sources orales, viennent éclairer la richesse et la diversité d’expériences fortement marqués par leur contexte singulier. Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. This is an Open Access article distributed under the terms of the prevailing CC-BY-NC license at the time of publication. http://creativecommons.org/licenses/by-sa/4.0 Downloaded from Brill.com 10/24/2024 04:03:06AM via Open Access. 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https://openalex.org/W2568478230	https://journal.ugm.ac.id/jkap/article/download/12557/11340	English	null	An Analysis of Poverty Reduction Program Based on the Conditional Cash Transfer (CCT) (A Case of the Family Hope Program Implementation in Pandak Bantul District 2014)	JKAP (Jurnal Kebijakan dan Administrasi Publik)/JKAP. Jurnal Kebijakan dan Administrasi Publik	2,016	cc-by-sa	6,068	Abstract The study of this research was an analysis of the public policy implementation concerning on the implementation of the Family Hope Program (PKH) in Pandak, Bantul regency in 2014. The pur- poses of this study were to investigate: (1) the implementation of the Family Hope Program (PKH), (2) the performance of the Family Hope Program (PKH) and (3) factors that influence the perfor- mance of the Family Hope Program (PKH). To investigate the implementation of the Family Hope Program in Pandak, the researcher tried to analyze the processes during its implementation. While to measure the performance of the Family Hope Program in Pandak, the researcher applied policy output indicators approach from Randall B. Ripley. It consists of indicators of access, scope, ac- countability, be as, promptness of service and suitability of the program needs. The research used a qualitative method by using primary data and secondary data. To collect the data, the researcher used observation, interview and documentary. To analyze the data, the researcher applied induc- tive data analysis. PKH implementation consists of some the steps, those are: (1) determining the targets, (2) validating and preparing of the initial meeting, (3) the distributing of aid and clustering the participants of PKH,(4) commitments verification, and (5) updating the data. From the various stages taken, there is a problem related to the weaknesses of the validation process, that it is only administratively. The weak validation process leads the determination of the target program be- came less accurate. Based on the measurement of the policy output indicators showed that the output performance of the Family Hope Program (PKH) in Pandak is low, it can be seen from: 1) aspect of bias, as there are still economically, some established families belonging to the mem- bers of PKH; 2) aspects of delivery service (promptness of service) as the lateness of the financial aid disbursement, and 3) aspects of the sustainability of the program with the needs of the target group, the aid criteria or the amount of aid considered to be unfair for each RTSM. JKAP (Jurnal Kebijakan dan Administrasi Publik) Vol. 20 No 1 – May 2016, p56-67 p-ISSN 0852-9213, e-ISSN 2477-4693 Available Online at http://journal.ugm.ac.id/jkap Received: March 2016 Accepted: April 2016 Received: March 2016 Accepted: April 2016 Received: March 2016 Accepted: April 2016 Published: INTRODUCTION such as education and health. In Indonesia the Family Hope Program was first implemented in 2007 piloting in seven provinces (West Sumatra, Jakarta, West Java, East Java, East Nusa Tengga- ra, North Sulawesi and Gorontalo). As in DIY, including Bantul, PKH was started in 2008. Poverty is a global social problem. Both developing and developed country can not be separated from the problem. Indonesia as a de- veloping country is also facing the poverty prob- lem. Policies and poverty alleviation programs has been actually done by the government re- peatedly. However, it can be said that the pov- erty alleviation program that have been executed was not effective in solving the poverty problem. Although in Bantul and Yogyakarta district PKH has been started in 2008, but up to 2013, the poverty rate in Yogyakarta precisely reached 15.03 percent. This amount is still larger than the average of the national poverty rate, that is 11.47 percent. Besides, PKH which has been running in Bantul District since 2008 found sev- eral problems that there are some people of both PKH receivers and non-receivers who do not un- derstand what PKH is. Moreover, there are some PKH receivers in educational case who violates the presence commitment at least 85% in school effective day (http://bbppksjogja.depsos.go.id/). Pandak belongs to poverty area in Bantul Dis- trict. Besides, Pandak sub-district has the highest number of PKH participants compared to other sub-district. That is why the associated problems of the target accuracy, the coordination of the implementation, and the appropriateness of the policy objectives are becoming more complex and interested to be researched. Indonesian government still continues to improve some policies and programs to alleviate the poverty problems. One of them is the issu- ance of the Indonesian Government Regulation No. 15 Year 2010 on accelerating poverty allevi- ation. Based on the regulation, the government formed a national team to accelerate the allevia- tion of poverty (TNP2K). The government has currently an integrated poverty alleviation pro- grams, those are the poverty reduction programs based on a social assistance, the poverty reduc- tion programs based on a community empower- ment and the poverty reduction programs based on the empowerment of small businesses (http:// www.tnp2k.go.id/id/mengenai-tnp2k/tentang- tnp2k/ downloaded on 27/05/2015). The poverty alleviation programs based on the social assistance is a new term in Indonesia. Abstract g p f f f The research results also showed five dominant factors toward the performance of the program, namely: 1) the lack of communication with the local village government; 2) the lack of data trans- parency of the PKH receiver targets; 3) the limitation of the available sources; and 4) the absence of mechanisms and rules controlling the fund spent by RTSM; and 5) the decreased compliance of RTSM in fulfilling the obligations under the rules of the program. According to these factors, there were some recommendations to increase the policy / program to be better, those are: (1) Improving the communication and involving the local village government, (2) Increasing the data transparen- cy of the PKH receiver targets; (3) there should be an audit of the available sources; (4) Social Ministry should issue a new rule and control it to the spending of PKH financial aid by RTSM, and (5) optimizing the functions and the coordination between the supervisors and the local village government to increase the awareness of the poor society in fulfilling the specified obligations in the program. Keywords: implementation of the program, the performance of the program, the family hope pro- gram y gram Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 JKAP (Jurnal Kebijakan dan Administrasi Publik) Vol.20, No 1 May 2016 ---- http://journal.ugm.ac.id/jkap INTRODUCTION Ripley (1985), which can be explained as follows: - 1) Access: to know that the program or service provided is easy to reach and those who responsible in imple- menting the policies or programs are easy contacted by the target group; 2) Coverage: to assess the extent of the target groups can be reached by public policy; 3) Frequency: it is used to measure how often the target group can obtain the ser- vices of a policy or a program; 4) Bias: to assess whether the services provided by the holder or the government deviant from the non-target group or the non-eligible target group to get the services provided through a policy or program; 5) Service delivery (service accuracy): to assess whether the ser- vices provided in the implementation of a program is done on time or not.; 6) Accountability: to assess whether the holder or the government action in carry- ing out their duty to deliver the output to the target group policy can be justified; 7) Com- pliance program needs: used to measure whether the policy or program output re- ceived by the target group is consistent with their needs or not. Indicators policy outcomes: The second indi- cator is the policy outcomes. It is applied to scribed by Dunn (2003: 21) that formulation of the problem is defined as an effort to generate information about the conditions that causes pol- icy problems. The stages in forming the appro- priate policies can be explained suited with Dunn theory (2003: 24-25). Generally the stages are as follows: 1) Composing the agenda; 2) Policies Formation; 3) Policies Adoption4) Policies Im- plementation and, 5) Policies Evaluation. From the discussion above, related to the definition of policies expressed by the experts, the author concluded that the public policy is a series of actions and decisions made by the government that has purposes to solve the public problems. Therefore, in this study, PKH is the main subject of the government policies or programs, which the product of the policies for- mulation was established by the Social Ministry that finally becomes Family Hope Program (PKH). The implementation is one step of the pub- lic policy processes after the policy formulation stage. Nugroho (2014) states that the policy im- plementation is principaly a way to achieve a policy goal. INTRODUCTION It is a program providing conditional grants or better known as the Conditional Cash Trans- fer (CCT). The main characteristics of the CCT program is requiring an attitude which should be done by the program receivers. Family Hope Program (PKH) is Conditional Cash Trans- fer (CCT), which is entered into cluster 1, where the social assistance and the protection programs are aimed in fulfilling the basic human rights According to Carl Frederick in Wahab (1997: 3) policy is an action that leads to the goal proposed by a group or government in the partic- ular environment connected with certain obsta- cles while seeking some opportunities to achieve the desired goals or objectives. Whereas, seen as a process, public policy consists of three (3) main dimensions, namely (1) formulation, (2) implementation, and (3) evaluation. The initial stage of the public policy is composed based on the existing formulation of the problem, as de- 57 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... target group. It covers the presence of grant activity distribution, subsidies, and the like. They were all carried out in the implementa- tion of a policy. There are various indicators that can be used to assess the quality of the policy output which is adopted as the thought of Randall B. INTRODUCTION The implementation that is associat- ed with the policy is not only formulated and then stated in the form of legislation but also is implemented in order to reach the desired effects or purposes. Purwanto and Sulistyastuti (2012: 106- 110) state that the main indicator in measuring the performance of the implementation can be divided into two. They are the output indicator policy (policy output) and the outcome indicators policy (policy outcomes). It will be presented as follows : b. Indicators policy outcomes: The second indi- cator is the policy outcomes. It is applied to assess the results of the policy implementa- tion. In the literature, the outcome indicator is also referred as an indicator of the impact of a. Policy indicator output is used to determine the direct consequences that can be felt by the a. Policy indicator output is used to determine the direct consequences that can be felt by the 58 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 JKAP (Jurnal Kebijakan dan Administrasi Publik) Vol.20, No 1 May 2016 ---- http://journal.ugm.ac.id/jkap Output indicators Further Analysis - Access - RTSM participants can obtain services easily - RTSM participants meet service providers easlily - Reducing the compliance of the RTSM - Scope - The magnitude of the reach of the target group - Accountability - Accountability of the officer - Whether the RTSM rights reduced or not - Refraction - Appropriateness of granting program to the target group - Service delivery - Timeliness of assistance - Conformity program with needs - Fitness for purpose pogram needs RTSM Table. 1. The formulation of the policy output indicator in assessing the performance of the implementation of CCT Pandak Table. 1. The formulation of the policy output indicator in assessing the performance of the implementation of CCT Pandak the policy (policy impact). In fact, formulat- ing the impact indicator is not that easy to do. It is caused by two things: 1) the extent of the policy coverage; 2) The unspecific policy objectives. Since both of these policies ware so abstract and broad that they are not easy to specifically mention how broad the scope of the actual policies (education, health, agricul- ture, etc.) is. Table. 1. There are several theories explaining the factors influencing the implementation of pro- grams / policies (Table 2). Starting from several factors affected the implementation of a policy or a program that has been described by experts above. In this study, researchers plot some sus- pected factors affecting the implementation of the Family Hope Program in Pandak, namely: 1) Communication 2) Resources; 3) Commitment; and 4) Support of Target group. In this context, to get the valid measure- ment of the performance of the policy implemen- tation, it will only focuses on the formulation of some selected indicators of the selected policy output to be analyzed. It is because of the policy Family Hope Program in Pandak get a saturation or an addition of participants from 2014 until now. Today, they are still in the implementation process.Therefore it is still too early to evaluate the impacts of the policy. This is in line with the opinion of Indiahono (2009: 143) that the policy outcomes are usually measured after the output release or within a long time of post- implementation. Policy formulation of the output indicator in this study can be seen in This study examines the stages of the im- plementation process of the Family Hope Pro- gram in Pandak. It is expected that the suitability of the Family Hope Program implementation in Pandak under the regulations / guidelines of the implementation of the general program can be explained clearly. Furthermore, to measure how a policy/a program works and reaches the expec- tations, this study used policy output indicators based on the approach proposed by Randal B. Ripley (1985), which are: (1) Access: how RTSM can access or reach PKH; (2) Scope: how 59 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... Name Factors Edward III Communication Resource Disposition Bureaucratic structure Merilee S Grindle Contents policy Implementation environment Daniel A Mazmanian and Sa- batier Characteristics of the problem Characteristics policies Environment variables Donald S Van Meter and Carl E. Table. 1. Van Horn Standard and policy targets Resource Communication between the organization and the strengthening of activity Characteristics of the implementing agency Social conditions of political and economic Disposition implementor G Shabbir Cheema and Den- nis A Rondinelli Environmental conditions Relations between organizations Resource organization for policy implementation Characteristics and capabilities of the implementing agencies Donald P Warwick Political Leadership Commitment Organizational capabilities Commitments The Executor Support group interests Table 2. Factors that affect the process of the policy implementation Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... Table 2. Factors that affect the process of the policy implementation good RTSM’s opportunity in reaching out the overall programs in Pan- dak; (3) Accountability: the officers’ responsi- bility and the suitability of the targets’ rights (4) Bias / Distortion: whether there are benefi- ciaries from not-very-poor households or not; (5) The accuracy of the service (service delivery): the time efficiency of the PKH aid delivery to RTSM; and (6) Suitability of the program requirements; how far PKH can meet the RSTM needs in accessing educa- tional and medical services (Figure 1). municated to the stakeholders, the target group of the policy, and any other related parts. The communication can be so either directly or indi- rectly that the policy objectives can be achieved effectively and efficiently. In this study, the communication seen from some aspects which are among PKH executors, the executor with PKH participants, and the communication be- tween the PKH executors with the local village government; (2) resource, it is an input of the PKH taken from the aspect of human resources of UPPKH Bantul, the supervisors, and the exist- ing infrastructures which are used in the imple- mentation of the PKH; (3) Commitment, it is the seriousness and sincerity from all program executors in successing the program. In this The possible factors that influence the im- plementation performances are: (1) communica- tion, public policies information need to be com- 60 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 JKAP (Jurnal Kebijakan dan Administrasi Publik) Vol.20, No 1 May 2016 ---- http://journal.ugm.ac.id/jk Figure 1. Policy output indicators proposed by Randal B. Ripley Figure 1. Policy output indicators proposed by Randal B. Ripley in Bantul and in DIY. Second, the presence of the saturation / additions of the participants which started to be implemented in 2014. Table. 1. Third, in the implementation of PKH, it is still found a variety of problems, e.g : the validation process was not so appropriate with the rules that the val- idation results are not accurate, the verification process on the participants' commitment on health and education was not optimal, in educa- tion it is still found that the participants’ children violate the commitment of a minimum of 85% attendance of school effective days. study, the commitment means the seriousness of the local government and the executors of the program in successing of the program; (4) Support Target group, in the context of this study, it focuses on the understanding of the CCT target group and their compliance toward the program rules. RESEARCH METHODS At this stage, the re- searchers first transcribe the interviews and the observation. They then adapt into a varie- ty of the secondary data from the documents obtained. The election results were then cate- gorized to determine the aspect that was as- sessed against the specified domain, namely the process of implementation of the Family Hope Program in Pandak. 1. Data reduction is the sort phase of relevant primary and secondary data to the object studied in the research. At this stage, the re- searchers first transcribe the interviews and the observation. They then adapt into a varie- ty of the secondary data from the documents obtained. The election results were then cate- gorized to determine the aspect that was as- sessed against the specified domain, namely the process of implementation of the Family Hope Program in Pandak. 1. The informant is a person who is supposed to really know the phenomenon that became the object of research. They can help the au- thor in exploring the information and the data required in a qualitative approach. Informants can be selected intentionally (purposive) as a sample. It is determined by the author in ac- cordance with the purpose of the research (Moleong, 2004: 157). 2. Document, a written materials or objects re- lated to a specific event or activity (Moleong, 2007: 159). In this research, documents that are used by the author are guidelines books of PKH, the report documents of the implemen- tation of the CCT and other offi- cial documents. 2. Presentation of Data, it is claimed as a struc- tured collection of information that gives the possibility of drawing conclusions and taking actions related to the implementation of the program. Presentation of the data in this re- search is conducted by using tables, pictures and a series of sentences arranged so coher- ently that the writer can draw the conclusions accurately. 2. Presentation of Data, it is claimed as a struc- tured collection of information that gives the possibility of drawing conclusions and taking actions related to the implementation of the program. Presentation of the data in this re- search is conducted by using tables, pictures and a series of sentences arranged so coher- ently that the writer can draw the conclusions accurately. RESEARCH METHODS This study is a qualitative descriptive study and it involves case study approach. The subjects of this study are individuals, groups, institutions and communities. A case study approach is used because the characteristics of this research are specific, special, and local scale that are associat- ed with the implementation of the CCT in Pan- dak, Bantul. To determine the extent of the PKH imple- mentation and utilization by UPPKH, supervi- sors, local village government, and RTSM tar- gets, the data are collected in the form of expla- nation and experiences from UPPKH, supervi- sors, and village officials. The RTSM knowledge about the stages of the implementation of the Family Hope Program is also identified especial- ly with the extend of saturation which were im- plemented in 2014 in Pandak. To explore the wide range of the relevant information, descrip- This study took place in Pandak, Ban- tul. The reasons why this research took place in Pandak are: first, this district is a point pockets of poverty that exists in Bantul and Yogyakar- ta. Second, this district is an area that has the biggest target of the Family Hope Program, both 61 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... tive qualitative research techniques are applied in collecting the information on the Family Hope Program implementation, determining the per- formance of the program implementation, and finding the factors influencing the implementa- tion of the Family Hope Program. port interview toward the goal of the target groups; 4) Library, it is administered to get vari- ous kinds of books (reference) to select concepts, theories and notions needed as a basic theory in this study. Analysis of the data used in this research is the analysis of data developed by Miles and Hubberman (1992: 16-20). It uses an interactive model analysis with the stages of the data reduc- tion, the data presentation and the conclusions drawing (verification) which are explained as below: According to Lofland and Lofland cited in Moleong (2007: 157), the primary data sources in qualitative research are the words, actions, and the rest are additional data such as documents and others. Moreover, the source of the data used can be described as follows: 1. Data reduction is the sort phase of relevant primary and secondary data to the object studied in the research. RESEARCH METHODS The main techniques used for the data col- lection in this study are as follows: 1) In-depth interviews (in-depth inteer- views) were conducted to find out the topic deep- ly; 2) Observation, it is aimed in revealing infor- mation that cannot be accommodated through the interviews; 3) Documentation, it is the collection of documents used in addition to a basic constit- uent of interview materials and tools to sup- 3. Drawing conclusions (Verification), it is a rethinking stage of the activities that came to the writer’s mind during taking notes or re- viewing. It is completely the final process of the data analysis process through interpreta- tion tendencies according to the results of da- 3. Drawing conclusions (Verification), it is a rethinking stage of the activities that came to the writer’s mind during taking notes or re- viewing. It is completely the final process of the data analysis process through interpreta- tion tendencies according to the results of da- 62 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 62 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 AP (Jurnal Kebijakan dan Administrasi Publik) Vol.20, No 1 May 2016 ---- http://journal.ugm.ac.id/jkap NO VILLAGE Total RTSM 1 Caturharjo 389 2 Gilangharjo 603 3 Triharjo 628 4 Wijirejo 267 DISTRICTS 1887 Table 3. Composition RTSM PKH participants in Pandak 2014 Source: UPPKH Bantul 2014 Table 3. Composition RTSM PKH participants in Pandak 2014 Source: UPPKH Bantul 2014 RESULT AND DISCUSSION ta reduction based on the theoretical frame- work. The conclusion is drawn by providing a description of the possible implications on the findings of the aspects presented. Implementation of the Family Hope Program Implementation of the Family Hope Pro- gram (PKH) in Bantul has been started since 2008 until 2014. The program is still run- ning. Whereas, in 2009 Pandak was included in the CCT program for the first time with 500 RTSM. However, as the time goes on, there is a saturation or an addition of the participants number of PKH adjusted in the Data Collection of Social Protection Program (PPLS) in 2011. Therefore, at the end of 2013 there were CCT target additions in Bantul district with a very significant increasing number compared to the previous years. The significant addition also occurs in Pandak which initially only co- vers 389 RTSM at the beginning of the year 2013 to 1.887 at the beginning of 2014. The da- ta composition of RTSM per-village in the Pan- dak district can be seen in Table 3. It shows that the RSTM total number in Pandak in early 2014 is as many as 1,887 RTSM. Triharjo village is a village in Pandak which has the the highest RTSM for about 628. Meanwhile, Wijirejo vil- lage has the smallest coverage, that is 267 RTSM. To get data validation, this study applied the data triangulation technique. Triangulation can be defined as a data collection technique combining various data collection techniques and data sources that already exist (Sugiyono, 2009: 83). The phases that should be passed in the triangulation technique are: 1) Conducting depth interviews with informants; 2) Doing the crossing test between information obtained from informants with the results of the re- search; 3) Performing confirmation of the results obtained to the other informants or oth- er sources. In connection with this research on the implementation of the CCT in Pandak, then the writer should compare the opinions and the views among the PKH participants community in Pandak, Associate CCT Pandak, UPPKH, PKH teamwork in Bantul District Social Service and the community / village in the region Pandak to check the validity of the data. 63 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... Commentary Table 4. The Analysis of The Implementation Phase of PKH Table 4. The Analysis of The Implementation Phase of PKH mentation of the Family Hope Program in Pan- dak can be seen in Table 5. The implementation performance of CCT in Pandak was still ineffec- tive if it is seen from the three output indica- tors, namely: Bias, Accuracy of Time Services and Compliance between Needs and Pro- gram. Based on the bias indicators, the imple- mentation of CCT in Pandak was still covered by the extence of established households economi- cally, that still become PKH participants. Mean- while, according to yhe time accuracy of service, it was found that the disbursement of PKH financial assistance was often not in time The research results on the stages of the implementation of the CCT Pandak can be seen in Table 4. It can be observed that the weakness- es of the implementation process of CCT in Pan- dak occurred due to the absence of prospective participant field validation PKH. With the ab- sence of the field validation toward the PKH par- ticipants, inaccuracy of PKH participants target may appears. The performance of the Family Hope Pro- gram implementation Implementation of the Family Hope Program Stages of PKH Implementation Commentary Goal Setting Process (Targeting) Pandak was worthy of being PKH target because: 1) the local government’s commitment to Bantul, 2) high poverty rate, 3) indicates malnutrition and transition rates from pri- mary to secondary school and, 4) the availability of infra- structure (supply) both education and health The Validation Process to get Ini- tial Determination PKH partici- pants There was no validation in th real condition of RTSM PKH recipients in the field. Validation was done only adminis- tratively and it was possible to be misplaced. Disbursement Process UPPKH delay occured in providing and distributing PKH membership cards. However, the distribution of aid could still be realized. Yet their membership card could be over- comed by an evidence of KK invitation and a copy of each RTSM participants. Commitment Verification Commitment verification could be both conducted and ex- ecuted well monthly. Data Update Updating data process run well, because the companion always actively controlled the data changes, either through monthly meetings with the Chairman of the Group or through a SIM data. Table 4. The Analysis of The Implementation Phase of PKH Stages of PKH Implementation Stages of PKH Implementation The performance of the Family Hope Pro- gram implementation In summary, the performance of the imple- 64 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 AP (Jurnal Kebijakan dan Administrasi Publik) Vol.20, No 1 May 2016 ---- http://journal.ugm.ac.id/jkap Output Indicators Effective Ineffective Access √ - Scope √ - Refraction - √ Accountability √ - Timeliness of Service - √ Conformity Program Needs - √ Table 5. The Analysis of Output Indicators of Implementation Performance in CCT Pandak e 5. The Analysis of Output Indicators of Implementation Performance in CCT Pandak and likely to be delayed. Meanwhile, based on the compliance between needs and program in- dicators, the financial aid for RTSM can not be synchronized with the RSTM needs in education and health as the plan. The amount of the aid which is implemented does not reflect the fair- ness. Further, the disbursed aid often creates a gap among RTSM participants with the different member composition of the house stairs and the different education charge. the comparisson between the number of human resources of the PKH implementers and the lim- ited and ineffective facilities in supporting the implementation of the CCT; 3) commitments, it includes the commitment of Bantul District Gov- ernment and the implementers as well that has ben already good and effective in supporting the implementation of CCT; 4) Support from the tar- get group includes; good understanding about PKH from the target group. However it has not been followed by the RSTM compliance to the provisions of the program because there is no rules to report the spend of funds by RTSM. The commitment to alleviete violations in the areas of education and health shoud be increase. The factors affecting the implementation of the Family Hope Program Factors that affect the implementation of the Family Hope Program in Pandak District, as presented in Table 6, can be seen that the im- plementation of the CCT Pandak is influenced by: 1) Communication, consists of communica- tion among the effective staff implementers, communication between the implementers and the effective RTSM , but in fact, the communica- tion between implementers and the local village government was not very effective. Village Gov- ernment claimed that the CCT is a closed pro- gram due to lack of relevant detail transparency of PKH target. Besides, the village government had also never been involved in the implementa- tion of the CCT coordination; 2) Resources, it is CONCLUSIONS Based on the data analysis that have been described in the previous sections, it can be con- cluded as follows: 1. Implementation of the Family Hope Pro- gram in Pandak in 2014 covered 1887 Very Poor Households (RTSM) partici- pants of PKH. From the various stages that carried out, there are still some problems found. It is associated with the validation process which was only conducted admin- 65 6 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 Ahmad Hanif – An Analysis of Poverty Reduction Program Based on the Conditional Cash..... Factors Implementation Effective Ineffective Communication Communication among the staffs √ - Communication between the executives and RTSM participants √ - Implementing communication with the Village - √ Resource Total Executive - √ Means and Prasana Support - √ Commitment Regional commitment √ - commitment Implementation √ - Support Target group Kelompok Goal √ - Compliance Target group - √ Table 6. Factors that affect the implementation of the Family Hope Program Factors Implementation Effective Ineffective Communication Communication among the staffs √ - Communication between the executives and RTSM participants √ - Implementing communication with the Village - √ Resource Total Executive - √ Means and Prasana Support - √ Commitment Regional commitment √ - commitment Implementation √ - Support Target group Kelompok Goal √ - Compliance Target group - √ Table 6. Factors that affect the implementation of the Family Hope Program Table 6. Factors that affect the implementation of the Family Hope Program istratively without being followed by di- rect/field inspection of the prospective par- ticipants PKH that may leads less accurate targets. ity of resources in the form of supervisors and limited appropriate infrastructures, 4) the absence of mechanisms and rules the require the participants to report the spend of funds of RTSM program, and 5) the re- ducing of the obedience of RTSM partici- pants in fulfilling obligations under the rules of the program. 2. Performance of PKH implementation in Pandak District cannot be said to be com- pletely good. CONCLUSIONS It is based on the assessment of the implementation performance indica- tors, namely: 1) bias aspects, there are still some established households that became PKH participants; 2) aspects of service de- livery (promptness of service), the dis- bursement of the financial aid is often not in time; 3) aspects of the conformity of the program with the needs of the target group, the aid rules and the amount of the aid are felt to be unfair for each RTSM. It is claimed that those have not been able to meet the expected needs. 2. REFERENCES Dunn, W., 2003. Pengantar Analisis Kebijakan Publik. Gadjah Mada University Press. Yogyakarta. Indiahono, Dwiyanto. 2009. Kebijakan Pub- lik :Berbasis Dynamic Policy Analisys. Gava Media. Yogyakarta. Knill, Christoph dan Jele Tosun. 2012. Public Policy: A New Introduction. New York: Palgrave Macmillan. g Koentjoroningrat. 1997. Metode-Metode Penelitian Masyarakat. PT. Gramedia. Ja- karta Kusumanegra, Solahuddin, 2010. Model dan Aktor dalam Proses Kebijakan. Penerbit Gava Media. Yogyakarta Performances of the Family Hope Program Implementation are influenced by: 1) the lack of communication to the village gov- ernment, 2) the lack of the data transparen- cy of the PKH recipients, 3) the availabil- 3. Kraft, Michael dan Scott. R. Furlong. 2010. Pub- licPolicy: Politics, analysis, and Alterna- tive. USA: CQ Press. 66 Copyright © 2016, JKAP, p-ISSN 0852-9213, e-ISSN 2477-4693 (Jurnal Kebijakan dan Administrasi Publik) Vol.20, No 1 May 2016 ---- http://journal.ugm.ac.id/jkap Miles dan Huberman, 1992. Analisa Data Kuali- tatif. PenerbitUniversitas Indonesia. Ja- karta Lalu, F. 2014. Kinerja Implementasi Kebijakan Penanganan Perempuan Korban Kekera- san. JKAP. Magister Administrasi Publik, Universitas Gadjah Mada. Yogyakarta Volume 18, Nomor 2 Moleong, Lexi J. 2006. Metodologi Penelitian Kualitatif (EdisiRevisi). Penerbit Rosda- karya. Bandung Nainggolan,T,dkk. 2012. 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Yogyakarta Volume 18, No- mor 2 Parsons, Wayne. 2008. Public Policy, Pengantar Teori dan Praktik Analisis Kebijakan. Kencana Prenada Media Group. Jakarta Purwanto, AE dan Sulistyastuti, DR. 2012. Im- plementasi Kebijakan Publik: Konsep dan Aplikasinya di Indonesia. Penerbit Gava Media. Yogyakarta Rawlings, L. & Rubio, G. 2005.Evaluatingthe impact of conditional cash transfer pro- grams. The World Bank Research Remi, Sutyastie Soemitrodan Prijono Tjiptoher- ijanto. 2002. Kemiskinan dan Ketidakmer- ataan di Indonesia. Rineka Cipta. Jakarta Sitompul, Mukti. 2006. REFERENCES ‘Implementasi Kebijakan Publik: Pengalaman Masa Lalu’Jurnal Harmoni Sosial, Vol. 1 No. 1, FISIP Uni- versitas Sumatera Utara. Subarsono, AG, 2006. Analisis Kebijakan Pub- lik: Konsep, Teori dan Aplikasi. Pustaka Pelajar. Yogyakarta Direktorat JaminanSosial.(2013), Naskah Pe- doman Umum Program Keluarga Harapan. Jakarta: Kementerian Sosial RI Sugiono. 2009. Memahami Penelitian Kualitatif. Alfabeta. 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https://openalex.org/W2056579837	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0053502&type=printable	English	null	A New Test of Attention in Listening (TAIL) Predicts Auditory Performance	PloS one	2,012	cc-by	10,382	A New Test of Attention in Listening (TAIL) Predicts Auditory Performance Yu-Xuan Zhang1,2, Johanna G. Barry1, David R. Moore1, Sygal Amitay1 1 Medical Research Council - Institute of Hearing Research, Nottingham, United Kingdom, 2 National Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China Xuan Zhang1,2, Johanna G. Barry1, David R. Moore1, Sygal Amitay1 Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: yuxuan.zhang@ihr.mrc.ac.uk on a subset of stimuli or stimulus features (task relevant dimensions) among all that are present. For example, tone frequency discrimination requires judgments to be made based on tone pitch. Other aspects of the stimuli including level, duration, and location are not useful for successful task perfor- mance and are regarded as task irrelevant dimensions. In everyday life, we are constantly assigning and switching listening priority between different sound sources or features (e.g., voices [13]). Thus, the ability to select and focus on task relevant dimensions may contribute significantly to perceptual performance in addition to perceptual acuity. We developed a Test of Attention in Listening (TAIL) as the first step towards identifying and quantifying such contributions. Abstract Attention modulates auditory perception, but there are currently no simple tests that specifically quantify this modulation. To fill the gap, we developed a new, easy-to-use test of attention in listening (TAIL) based on reaction time. On each trial, two clearly audible tones were presented sequentially, either at the same or different ears. The frequency of the tones was also either the same or different (by at least two critical bands). When the task required same/different frequency judgments, presentation at the same ear significantly speeded responses and reduced errors. A same/different ear (location) judgment was likewise facilitated by keeping tone frequency constant. Perception was thus influenced by involuntary orienting of attention along the task-irrelevant dimension. When information in the two stimulus dimensions were congruent (same-frequency same-ear, or different-frequency different-ear), response was faster and more accurate than when they were incongruent (same-frequency different-ear, or different-frequency same-ear), suggesting the involvement of executive control to resolve conflicts. In total, the TAIL yielded five independent outcome measures: (1) baseline reaction time, indicating information processing efficiency, (2) involuntary orienting of attention to frequency and (3) location, and (4) conflict resolution for frequency and (5) location. Processing efficiency and conflict resolution accounted for up to 45% of individual variances in the low- and high-threshold variants of three psychoacoustic tasks assessing temporal and spectral processing. Involuntary orientation of attention to the irrelevant dimension did not correlate with perceptual performance on these tasks. Given that TAIL measures are unlikely to be limited by perceptual sensitivity, we suggest that the correlations reflect modulation of perceptual performance by attention. The TAIL thus has the power to identify and separate contributions of different components of attention to auditory perception. Received October 3, 2012; Accepted November 30, 2012; Published December 31, 2012 Received October 3, 2012; Accepted November 30, 2012; Published December 31, 2012 Copyright: 2012 Zhang 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 study was funded by the Medical Research Council (MRC), United Kingdom, through intramural funding to the MRC Institute of Hearing Research. All authors were MRC employees at the time the research was conducted. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org Introduction In auditory processing tasks, BM0 is Backward Masking with no silence gap between the target tone and the noise masker, BM50 is Backward Masking with a 50-ms silence gap between the target tone and the noise masker, SM is Simultaneous Masking with no spectral notch around the target tone, SMN is Simultaneous Masking with a spectral notch around the target tone, FD is Frequency Discrimination with a fixed standard tone, and FDR is Frequency Discrimination with a roving standard tone. doi:10.1371/journal.pone.0053502.g001 Figure 1. Schematic illustration of the Test of Attention in Listening (TAIL) and auditory processing tasks. For TAIL, Backward and Simultaneous Masking, one example trial of stimulus presentation is plotted. For Frequency Discrimination, two trials are plotted. Abbreviations: In TAIL, F stands for frequency, L for location, S for same, and D for different. In auditory processing tasks, BM0 is Backward Masking with no silence gap between the target tone and the noise masker, BM50 is Backward Masking with a 50-ms silence gap between the target tone and the noise masker, SM is Simultaneous Masking with no spectral notch around the target tone, SMN is Simultaneous Masking with a spectral notch around the target tone, FD is Frequency Discrimination with a fixed standard tone, and FDR is Frequency Discrimination with a roving standard tone. doi:10.1371/journal.pone.0053502.g001 dimension are used to avoid confound with perceptual difficulty. Other task-irrelevant dimensions (stimulus duration and level) are roved. in the TAIL is directed (‘oriented’) to the task relevant dimension via the orienting attention network. According to the Load Theory, under such a low perceptual load (one tone at a time), perceptual capacity is not exhausted and attention will ‘‘spill over’’ to task irrelevant information, allowing further processing of that information. Load Theory thus predicts involuntary orientation of attention to the distracting dimension in proportion to perceptual capacity spared from relevant information processing. In addition, perceptual processing of information in the distracting dimension may present conflicts to decision making, as the same/different relationship in the distracting dimension may be incongruent with that in the task relevant dimension. Resolution of response conflicts would be indexed by performance deterioration in the The design and interpretation of the TAIL were informed by and are consistent with the principles of attention embodied in the influential Attention System view [14,15] and the Load Theory of attention [16]. Introduction Auditory performance is determined by interactions of auditory sensation with attention, memory, vision, emotion and a variety of other, lesser influences [1,2,3,4]. Attention and memory have received much recent interest because they are particularly important for the assessment and rehabilitation of hearing difficulties [5,6,7]. The influence of attention has typically been demonstrated by examining how directing attention to and away from the target stimuli or stimulus features alters psychophysical (e.g., the dichotic listening paradigm; [8]) or physiological (e.g., hemodynamic signals [9], neuromagnetic fields [10]) measures of sound perception. Our goal here was to develop a behavioral test of auditory attention that can be used to identify and quantify the contribution of attention to auditory performance. TAIL measures the ability to focus selectively on a task relevant dimension and ignore information from task irrelevant dimensions using reaction time (RT) as the primary performance measure. In each trial, two clearly audible tones are presented sequentially (Fig. 1). The listener is asked to indicate whether the two tones are the same or different along one of two dimensions (frequency or location) as accurately and as quickly as possible. The other dimension is also systematically manipulated to serve as the distracting dimension. Highly distinctive variants of each stimulus Attention has been studied under a wide range of cognitive conditions and has so far eluded a consensual definition after over a century’s documented investigation [11]. At the core of most attention phenomena is the concept of selection: a subset of the available stimulus pool (including internal stimuli such as thoughts and memories) is examined more closely than and at the expense of others [12]. Most perceptual tasks also involve judgments based December 2012 \| Volume 7 \| Issue 12 \| e53502 December 2012 \| Volume 7 \| Issue 12 \| e53502 1 PLOS ONE \| www.plosone.org New Attention Test Predicts Hearing Figure 1. Schematic illustration of the Test of Attention in Listening (TAIL) and auditory processing tasks. For TAIL, Backward and Simultaneous Masking, one example trial of stimulus presentation is plotted. For Frequency Discrimination, two trials are plotted. Abbreviations: In TAIL, F stands for frequency, L for location, S for same, and D for different. December 2012 \| Volume 7 \| Issue 12 \| e53502 Introduction incongruent relative to the congruent case. Conflict resolution is typically used as a measure of executive control (e.g., the ’Stroop task’ [17,18]). Finally, when the tones are the same in both dimensions (without distracting or conflicting information), RTs provide a ‘baseline’ measure of information processing under minimum attention control. were systematically manipulated (Fig. 1). In the FL condition, frequency was the task-relevant dimension and location was the distracting dimension. Listeners were asked to press one of two buttons as accurately and as quickly as possible to indicate whether the two tones were the same or different in pitch. In the LF condition, location was the relevant and frequency the irrelevant dimension. Listeners chose whether the two tones were presented at the same or different ears. In the Control condition, neither frequency nor location was task relevant. Listeners were asked simply to press any button as soon as they heard the second tone. In all conditions, tone level was roved between 70 to 85 dB SPL and tone duration was roved between 100 and 300 ms. The silent gap between the two tones was fixed at 300 ms. Before the experiment, we ran pilot studies to ensure that variations within the roved ranges had no impact on the attention measures derived. We examined attention contribution to auditory performance by comparing RT measures of TAIL with threshold measures on three psychoacoustic tasks emphasizing spectral and temporal processing [5,19]: tone Frequency Discrimination, Backward Masking, and Simultaneous Masking. These tasks are recom- mended measures of ‘auditory processing’ (American Academy of Audiology, 2010) but may still be strongly influenced by attention skills [5]. For each task, we included a more demanding, high- threshold variant and a less demanding, low-threshold variant, in both of which threshold was assessed at 79% correct performance (Fig. 1). The stimuli used in these tasks were very simple, consisting of a single tone or a tone with a band-passed noise in each observation interval. Our current understanding of these tasks is not sufficient to make specific predictions about which attention components of the TAIL will contribute to performance on each task. However, some general trends can be predicted. First, these tasks showed differential activation of non-sensory cortical regions [20,21]. For example, Backward Masking produced greater activity than Simultaneous Masking in anterior cingulate cortex [21], a brain region critical for conflict monitoring and executive control [14]. Introduction The Attention System view suggests that attention is subserved by a system of brain networks that is neuroanatomi- cally separate from the information processing system (including stimulus encoding, analyzing, and decision making) and that this attention system consists of three separable networks carrying out, respectively, the functions of maintaining vigilance, orienting attention, and executive control. According to this view, attention December 2012 \| Volume 7 \| Issue 12 \| e53502 December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 2 New Attention Test Predicts Hearing were systematically manipulated (Fig. 1). In the FL condition, frequency was the task-relevant dimension and location was the distracting dimension. Listeners were asked to press one of two buttons as accurately and as quickly as possible to indicate whether the two tones were the same or different in pitch. In the LF condition, location was the relevant and frequency the irrelevant dimension. Listeners chose whether the two tones were presented at the same or different ears. In the Control condition, neither frequency nor location was task relevant. Listeners were asked simply to press any button as soon as they heard the second tone. In all conditions, tone level was roved between 70 to 85 dB SPL and tone duration was roved between 100 and 300 ms. The silent gap between the two tones was fixed at 300 ms. Before the experiment, we ran pilot studies to ensure that variations within the roved ranges had no impact on the attention measures derived. Four blocks of 40 trials were run for each condition, with the order of conditions randomized across listeners. Condition was switched between blocks and the corresponding instruction was displayed on the screen at the beginning of each block. Before the first block of each condition, a demo of 5 trials was used to familiarize the participants with the task. Each block of trials followed a two (same and different frequency) by two (same and different location) design. Tone frequencies were drawn randomly from the range 476–6188 Hz, with the constraint that the spectral gap between any two tones was at least 2.1 equivalent rectangular bandwidths (ERBs; [23]). This gap was well above the frequency discrimination thresholds of all of the participants and was intended to avoid perceptual confusion. The total twelve blocks of TAIL took about 20 minutes to complete. Equipment All testing was conducted in a sound attenuated booth on a PC, with all sounds delivered via circumaural headphones (Sennheiser HD 25). A USB-interfaced button box made in-house was used for response. The testing was fully automatic, with instructions displayed on the screen at the beginning of each block of trials. Auditory processing tasks. We assessed psychoacoustic performance on three tasks: Backward Masking, Simultaneous Masking, and Frequency Discrimination (Fig. 1). Briefly, a 3- interval, 3-alternative forced choice paradigm was used in all of the three tasks (for details, see [5]). At each trial, two identical, standard stimuli and one different, target stimulus were presented in random order. The listeners were asked to report the ‘‘odd-one out’’ by pressing a button. Across trials, the difference between the standard and the target stimuli was adaptively varied following a 3- Auditory processing tasks. We assessed psychoacoustic performance on three tasks: Backward Masking, Simultaneous Masking, and Frequency Discrimination (Fig. 1). Briefly, a 3- interval, 3-alternative forced choice paradigm was used in all of the three tasks (for details, see [5]). At each trial, two identical, standard stimuli and one different, target stimulus were presented in random order. The listeners were asked to report the ‘‘odd-one out’’ by pressing a button. Across trials, the difference between the standard and the target stimuli was adaptively varied following a 3- Introduction Thus, we expected differential patterns of attention contribution across the tasks. Second, as the simple stimuli are unlikely to exhaust perceptual capacity, we did not expect significant contribution of involuntary orientation. Third, and most critically, as the role of executive control is to facilitate focused attention onto the target information [14,22], we predicted that conflict resolution would more likely contribute to the high-threshold task variants in which the challenge of separating task relevant information from irrelevant information is greater. Performance on the lower threshold variant, in contrast, would more likely reflect the efficiency of the information processing systems themselves. Four blocks of 40 trials were run for each condition, with the order of conditions randomized across listeners. Condition was switched between blocks and the corresponding instruction was displayed on the screen at the beginning of each block. Before the first block of each condition, a demo of 5 trials was used to familiarize the participants with the task. Each block of trials followed a two (same and different frequency) by two (same and different location) design. Tone frequencies were drawn randomly from the range 476–6188 Hz, with the constraint that the spectral gap between any two tones was at least 2.1 equivalent rectangular bandwidths (ERBs; [23]). This gap was well above the frequency discrimination thresholds of all of the participants and was intended to avoid perceptual confusion. The total twelve blocks of TAIL took about 20 minutes to complete. Reaction times (RTs) on correct trials were used as the primary performance measure. RTs longer than 2 s or shorter than 200 ms, suggesting lapse of attention, interruption of performance or premature responding, were excluded (,0.8% trials). For the Control condition, the detection task allowed anticipated responses to the second tone and approximately 25% of RTs fell below the 200-ms criterion. We therefore analyzed the detection RTs with and without applying the low cutoff to check the impact of anticipated responses on the attention effects. For each of the FL and LF conditions, one (different) listener erroneously attended to the irrelevant dimension when incongruent information was presented. However, these listeners performed normally on congruent trials. Their data were excluded from the relevant analyses. Ethics Statement The research protocol was approved by the Nottingham University Hospitals Research Ethics Committee. All of the participants gave informed written consent and received an inconvenience allowance for their participation. For each TAIL condition, baseline RT was calculated using the trials on which the two tones were the same in both frequency and location. A two (same vs. different frequency) by two (same vs. different location) ANOVA with repeated measures was con- ducted on the RTs and the error rates. Involuntary orientation was indicated by the impact of the task irrelevant dimension(s) and quantified as the difference between the same and different trials for that dimension. Conflict resolution was indicated by the frequency by location interaction and was quantified as the difference between congruent (same or different in both dimen- sions) and incongruent (same in one dimension and different in the other) trials (Fig. 1). For each TAIL condition, baseline RT was calculated using the trials on which the two tones were the same in both frequency and location. A two (same vs. different frequency) by two (same vs. different location) ANOVA with repeated measures was con- ducted on the RTs and the error rates. Involuntary orientation was indicated by the impact of the task irrelevant dimension(s) and quantified as the difference between the same and different trials for that dimension. Conflict resolution was indicated by the frequency by location interaction and was quantified as the difference between congruent (same or different in both dimen- sions) and incongruent (same in one dimension and different in the other) trials (Fig. 1). Participants Nineteen volunteers between the age of 18 and 36 years (mean of 26 years, 9 females) were recruited from the University of Nottingham campus and nearby neighborhoods. All of the volunteers had 20 dB HL or better hearing thresholds for tones between 500 and 6000 Hz, bilaterally. Attention Effects in a Detection Task Frequency Discrimination was administered using the Psy- chtoolbox for Matlab, with 50 trials per condition. The masking tasks were administered using customized software (IHR-STAR [24]), with 20 trials per condition. Discrimination threshold (‘performance’) at 79% correct was evaluated by fitting psycho- metric functions using the maximum likelihood method imple- mented by the Psignifit toolbox for Matlab [25]. We examined whether the attention measures were contingent on the presence of a task relevant dimension by running TAIL in the Control condition, in which the task was simply to detect the second tone and neither frequency nor location was relevant (Fig. 5). RT was significantly shorter for different ear than for same ear presentation (F1,18 = 7.75, p = 0.01, gp 2 = 0.30). There was no significant effect of the relative frequency of the tones (F1,18 = 3.17, p = 0.09, gp 2 = 0.15) and no frequency by location interaction (F1,18 = 0.54, p = 0.47, gp 2 = 0.03). Because the detection task allowed anticipated responses due to the fixed inter-stimulus interval, we repeated these analyses excluding RTs shorter than 200 ms (,25% of all trials). The results remained the same, with a small significant but negative effect of location constancy (F1,18 = 5.39, p = 0.03, gp 2 = 0.23), but no effect of frequency constancy (F1,18 = 2.77, p = 0.11, gp 2 = 0.13) or congruency (F1,18 = 0.82, p = 0.38, gp 2 = 0.04). The significant negative effect of location constancy indicates that the second tone was processed and impacted responses. The lack of positive effects of feature constancy and dimension congruency suggest that they thus facilitated responses only when a certain stimulus dimension needed to be singled out for judgments. Task and Stimuli TAIL. TAIL was run in three conditions, in all of which tone frequency (F) and location (L; ear at which the tone was presented) PLOS ONE \| www.plosone.org December 2012 \| Volume 7 \| Issue 12 \| e53502 3 New Attention Test Predicts Hearing irrelevant frequency dimension did not significantly decrease the likelihood of making an error (F1,17 = 2.15, p = 0.16, gp 2 = 0.11). down/1-up staircase. For each task, two variants were run that yielded different levels of performance. For Backward Masking, the standard stimulus was a 300-ms bandpass noise (600–1400 Hz; 30 dB/Hz). The target stimulus had a 20-ms, 1-kHz tone preceding the noise, with the tone level starting at 90 dB SPL and varied adaptively. In the high-threshold variant (BM0), there was no gap between the tone offset and the noise onset; in the low- threshold variant (BM50), there was a 50-ms silent gap. For Simultaneous Masking, the same bandpass noise and 20-ms tone were used, but the tone started 200 ms after noise onset. In the high-threshold variant (SM), the noise was spectrally continuous; in the low-threshold variant (SMN), there was a spectral notch (800–1200 Hz) around the tone. For Frequency Discrimination, the stimuli were 100-ms tones presented at 75 dB SPL. In the low- threshold variant (FD), the standard tone frequency was fixed at 1 kHz; in the high-threshold variant (FDR), it was roved between 900 and 1100 Hz with a step size of 50 Hz [19]. TAIL Measures Directing attention to frequency. In the FL condition of TAIL, RT was shortest when the two tones were the same in both frequency and location (Fig. 2A), which we refer to as baseline RT. We conducted an ANOVA with repeated measures on RT with location (same vs. different ear) and frequency (same vs. different frequency) as within-subject factors. There was no difference between same and different frequencies [F1,17 = 2.54, p = 0.13, gp 2 (effect size) = 0.13], indicating approximately balanced processing for the two responses. RT was shorter for the same than for the different location (F1,17 = 41.98, p,0.001, gp 2 = 0.71), and shorter when the two dimensions were congruent (same or different in both dimensions, F1,17 = 33.60, p,0.001, gp 2 = 0.66) than when they were conflicting (same in one dimension but different in the other, Fig. 1). We thus identified two significant attention effects: Involuntary orientation to the task irrelevant dimension quantified as the RT difference between same- and different- location trials (in this FL condition), and Conflict resolution as the RT difference between congruent and incongruent trials (Fig. 2B). The involuntary orientation and conflict resolution effects in RT were mirrored by the error patterns (Fig. 2C, D). Error rate was higher for different- than for same-location trials (F1,17 = 25.36, p,0.001, gp 2 = 0.60) and for incongruent than for congruent trials (F1,17 = 21.35, p,0.001, gp 2 = 0.56). Relationship Among TAIL Measures We examined whether the TAIL measures were correlated, to determine whether they reflected separate functions. Though involuntary orientation and conflict resolution were present when attention was directed to either frequency or location, these measures were not significantly correlated either between or within stimulus dimensions (Table 1). In contrast, baseline RTs were correlated among all three conditions, despite the marked increase from 300 ms in the Control condition to approximately 500 ms in the FL and LF conditions (t.10, p,0.001, Cohen’s d .2.3). , p Directing attention to location. We examined whether the attention measures were dimension dependent by running the TAIL in a LF condition in which the task relevant and distracting dimensions were switched. RT analyses showed the same pattern of results as for the FL condition (Fig. 3). RT was longer for different than for same frequency, indicating involuntary orienta- tion of attention to the distracting dimension (F1,17 = 30.56, p,0.001, gp 2 = 0.64). Further, RT was longer for incongruent than for congruent trials, indicating a cost of conflict resolution (F1,17 = 95.12, p,0.001, gp 2 = 0.85). For error rate, only di- mension congruency had a modest but significant effect (F1,17 = 4.36, p = 0.05, gp 2 = 0.17), while constancy on the Effect of Testing Order We checked whether the attention effects resulted from confusion due to the mixed testing of the FL and LF conditions. If this were true, the effect for each condition would have emerged only after the other condition had been tested. For each condition, we compared the first and the last blocks of trials for those listeners who performed that condition first (Fig. 4). For both involuntary orientation and conflict resolution, RT and error rate differences on the first block were comparable to those on the last block (p.0.2), demonstrating that the effects were not caused by confusion of mixing the conditions. This result also showed the resistance of the attention effects to rapid learning caused by familiarization with the task set and testing environment. Left Ear Advantage? Here and in the following figures, error bars for RTs are within-subject SEM [50] and error bars for RT differences are across-subject SEM. doi:10.1371/journal.pone.0053502.g002 F1,17 = 8.4, p = 0.011, gp 2 = 0.32 for RT). A similar but lesser advantage was found in the RT for the Control condition (F1,17 = 4.7, p = 0.046, gp 2 = 0.23). However, in the FL condition, the effect of presentation ear on RT switched direction between the first and the second tones (interaction between ear and tone position, F1,17 = 8.9, p = 0.009, gp 2 = 0.36), with a left ear advantage for the first tone (follow up t test, t = 22.2, p = 0.042, Cohen’s d = 0.54) but a right ear advantage for the second tone (t = 2.6, p = 0.02, Cohen’s d = 0.63). Thus, the left-ear advantage for tonal stimuli appears to be modulated by attention, with the effect most prominent when attention is directed to location. F1,17 = 8.4, p = 0.011, gp 2 = 0.32 for RT). A similar but lesser advantage was found in the RT for the Control condition (F1,17 = 4.7, p = 0.046, gp 2 = 0.23). However, in the FL condition, the effect of presentation ear on RT switched direction between the first and the second tones (interaction between ear and tone position, F1,17 = 8.9, p = 0.009, gp 2 = 0.36), with a left ear advantage for the first tone (follow up t test, t = 22.2, p = 0.042, Cohen’s d = 0.54) but a right ear advantage for the second tone (t = 2.6, p = 0.02, Cohen’s d = 0.63). Thus, the left-ear advantage for tonal stimuli appears to be modulated by attention, with the effect most prominent when attention is directed to location. (orientation to frequency and location, conflict resolution for frequency and location, and baseline RT), only conflict resolution for frequency and baseline RT correlated significantly with threshold performance on the auditory processing tasks tested here (Fig. 6). Conflict resolution accounted for 45% of individual variance on FDR and 35% on BM0, but did not contribute significantly to SM (r = 20.084, p = 0.74) or to the easy conditions of the three tasks (p.0.05). Baseline RTs in the three TAIL conditions, in contrast, accounted for 43–47% of individual variance on BM50 and 43–46% on SMN. Left Ear Advantage? Finally, some studies have suggested a left-ear advantage for tonal stimuli [26,27,28]. Though any ear advantage should have been controlled for by the counterbalanced presentation of each tone at the two ears, we analyzed if such an advantage was present in the current study. In the LF condition, RT was facilitated when the sounds were presented to the left ear, both in terms of error rate (two ear by two tone position ANOVA, effect of ear, F1,17 = 13.9, p = 0.002, gp 2 = 0.69) and RT (F1,17 = 35.3, p,0.001, gp 2 = 0.47). This left-ear advantage was greater for the first tone than for the second tone (interaction between ear and tone position, F1,17 = 7.6, p = 0.014, gp 2 = 0.34 for error rate, PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org December 2012 \| Volume 7 \| Issue 12 \| e53502 4 New Attention Test Predicts Hearing Figure 2. TAIL performance for FL condition. A. Mean reaction time (RT) on correct trials. B. RT difference indicating involuntary orientation to location (RT [different location] – RT [same location]) and conflict resolution (RT [same in one dimension and different in the other] –RT [same or different in both dimensions]). C. Mean error rate. D. Error rate difference indicating involuntary orientation to location and conflict resolution. Here and in the following figures, error bars for RTs are within-subject SEM [50] and error bars for RT differences are across-subject SEM. doi:10.1371/journal.pone.0053502.g002 Figure 2. TAIL performance for FL condition. A. Mean reaction time (RT) on correct trials. B. RT difference indicating involuntary orientation to location (RT [different location] – RT [same location]) and conflict resolution (RT [same in one dimension and different in the other] –RT [same or diff i b h di i ]) C M D E diff i di i i l i i l i d fli l i H Figure 2. TAIL performance for FL condition. A. Mean reaction time (RT) on correct trials. B. RT difference indicating involuntary orientation to location (RT [different location] – RT [same location]) and conflict resolution (RT [same in one dimension and different in the other] –RT [same or different in both dimensions]). C. Mean error rate. D. Error rate difference indicating involuntary orientation to location and conflict resolution. TAIL Predicts Auditory Performance A primary motivation of the current study was to develop a test that could be used to assess the contributions of attention to auditory performance. As a first step towards this end, we examined here the extent to which the TAIL measures predicted individual variance in three auditory processing tasks. Among the five independent measures in the FL and LF conditions Left Ear Advantage? Note that all but one of the significant correlations in Fig. 6 would remain so even after a stringent Bonferroni correction for multiple comparisons (at the corrected alpha value of 0.008). PLOS ONE \| www.plosone.org Discussion The lack of correlation between the TAIL measures testified to the separation of the attention networks and information processing systems in audition, reminiscent of the Attention Network Test that was designed to test the Attention System view in vision [29]. In addition to integrating multiple facets of attention in one test, we demon- strated how these effects varied with task type (same/different discrimination versus detection) or with different task relevant dimensions (frequency and location). The TAIL measures revealed differential contributions of information processing and attention modulation to threshold performance on an array of psycho- acoustic tasks, with executive control accounting for up to 45% of individual differences in some high-threshold variants. To further illustrate the nature of the TAIL measures, we will compare each measure and the associated findings to the relevant attention literature. We will then discuss the implications of the attention contributions to auditory performance revealed by the TAIL. measures that could be mapped on to the Attention System proposal and the Load Theory of attention as 1) cost of resolving response conflicts, indexing the ability to focus selectively on the relevant dimension and ignore irrelevant ones in decision making, a function of the executive control networks, 2) involuntary orientation of attention to a task irrelevant dimension, reflecting spare perceptual capacity, and 3) response speed in the absence of distracting or conflicting information, indicating efficiency of the information processing system involved. The lack of correlation between the TAIL measures testified to the separation of the attention networks and information processing systems in audition, reminiscent of the Attention Network Test that was designed to test the Attention System view in vision [29]. In addition to integrating multiple facets of attention in one test, we demon- strated how these effects varied with task type (same/different discrimination versus detection) or with different task relevant dimensions (frequency and location). The TAIL measures revealed differential contributions of information processing and attention modulation to threshold performance on an array of psycho- acoustic tasks, with executive control accounting for up to 45% of Discussion The main goal of this study was to develop a test of attention in listening (TAIL) that could be used to identify contributions of attention to auditory performance. Through manipulation of task relevant and irrelevant dimensions, the TAIL yielded three PLOS ONE \| www.plosone.org December 2012 \| Volume 7 \| Issue 12 \| e53502 December 2012 \| Volume 7 \| Issue 12 \| e53502 5 New Attention Test Predicts Hearing Figure 3. TAIL performance for LF condition. A. Mean RT. B. RT difference indicating involuntary orientation to frequency (RT [different frequency] – RT [same frequency]) and conflict resolution (RT [same in one dimension and different in the other] – RT [same or different in both dimensions]). C. Mean error rate. D. Error rate difference indicating involuntary orientation to frequency and conflict resolution. doi:10.1371/journal.pone.0053502.g003 Figure 3. TAIL performance for LF condition. A. Mean RT. B. RT difference indicating involuntary orientation to frequency (RT [different frequency] – RT [same frequency]) and conflict resolution (RT [same in one dimension and different in the other] – RT [same or different in both dimensions]). C. Mean error rate. D. Error rate difference indicating involuntary orientation to frequency and conflict resolution. doi:10.1371/journal.pone.0053502.g003 Figure 3. TAIL performance for LF condition. A. Mean RT. B. RT difference indicating involuntary orientation to frequency (RT [differen Figure 3. TAIL performance for LF condition. A. Mean RT. B. RT difference indicating involuntary orientation to frequency (RT [different frequency] – RT [same frequency]) and conflict resolution (RT [same in one dimension and different in the other] – RT [same or different in both dimensions]). C. Mean error rate. D. Error rate difference indicating involuntary orientation to frequency and conflict resolution. doi:10.1371/journal.pone.0053502.g003 measures that could be mapped on to the Attention System proposal and the Load Theory of attention as 1) cost of resolving response conflicts, indexing the ability to focus selectively on the relevant dimension and ignore irrelevant ones in decision making, a function of the executive control networks, 2) involuntary orientation of attention to a task irrelevant dimension, reflecting spare perceptual capacity, and 3) response speed in the absence of distracting or conflicting information, indicating efficiency of the information processing system involved. Conflict Resolution The cost of resolving conflicts between sound frequency and location was used to assess the function of the executive control network. A classic example of conflict resolution measure is the Stroop task [17,18], in which a color name is presented in a physical color that matches or differs from that indicated by the semantic content of the word (e.g., the word ‘red’ presented in red or green font). Naming the color of the word was slower when in the differing (incongruent) than in the matching (congruent) condition, suggesting a cost of resolving the conflict between the two stimulus dimensions. The Stroop effect has been replicated using auditory verbal stimuli, in which the semantic content interfered with perception of pitch or loudness [30,31]. In vision, conflict resolution has also been demonstrated for non-verbal December 2012 \| Volume 7 \| Issue 12 \| e53502 December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 New Attention Test Predicts Hearing Figure 4. Comparison of RT (row 1 and 3) and error rate (row 2 and 4) measures between the n = 9) and the LF (B, n = 10) conditions in the listeners who performed that condition before doi:10.1371/journal.pone.0053502.g004 Figure 4. Comparison of RT (row 1 and 3) and error rate (row 2 and 4) measures between the first and fourth b n = 9) and the LF (B n = 10) conditions in the listeners who performed that condition before the other conditio Figure 4. Comparison of RT (row 1 and 3) and error rate (row 2 and 4) measures between the first and fourth blocks of the FL (A, n = 9) and the LF (B, n = 10) conditions in the listeners who performed that condition before the other condition. doi:10.1371/journal.pone.0053502.g004 December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 7 New Attention Test Predicts Hearing Figure 5. TAIL performance for Control condition. A. Mean RT to the second tone. B. RT gain as a function of frequency constancy, location constancy, and dimension congruency. doi:10.1371/journal.pone.0053502.g005 Figure 5. TAIL performance for Control condition. A. Mean RT to the second tone. B. RT gain as a function of frequency constancy, location constancy, and dimension congruency. doi:10.1371/journal.pone.0053502.g005 stimuli. Conflict Resolution Two widely used examples are the Simon task [32], in which congruency between stimulus location and response di- rection is manipulated, and the flanker task [29,33], in which conflicts are introduced in the spatial attributes of target and flanking stimuli. cruitment of executive control depends on task demand. In- congruent information in the two stimulus dimensions can be processed without interference and hence without the need for executive control unless the response is contingent on that information. This observation is consistent with the view of executive control as the ‘‘top-down’’ regulating signal conveying task demand [14]. g The TAIL provides a demonstration of non-verbal conflict resolution in the auditory domain. The previous Stroop-like tasks typically measure interference to or along a single stimulus dimension. For example, in the Stroop task, while word content interferes with color naming, there is little interference in the reverse direction. Asymmetric interference between different dimensions like this has been interpreted as reflecting the different degrees of automaticity in processing those dimensions [34]. In the case of the Stroop task, word reading has a more strongly established pathway than color naming in literate people, due to practice and daily use, and would be activated under less executive control (more automatic). In the TAIL, interference was demon- strated to occur in both directions between location and frequency, and to be of similar magnitude on the group level, but uncorrelated on the individual level. The TAIL results thus show that, on the group level, there was no difference in the automaticity of frequency and location processing, but individuals differed in the relative strength of the two pathways. Further, the conflict resolution effect disappeared when the task required simple detection of the second tone, indicating that the re- Involuntary Orientation According to the Load Theory of attention [16], involuntary orientation of attention to a task irrelevant dimension could be used to index the perceptual capacity spared from processing task relevant information for environment monitoring. While focusing on the task at hand requires exclusion of task irrelevant information, monitoring of the environment for potentially interesting events needs some attention to that information. The Load Theory suggests that, at a fixed cognitive load, attention to irrelevant information is determined by the perceptual capacity spared from task relevant processing. Thus, for a given task and stimulus set, the attention paid to task irrelevant information provides a measure of perceptual capacity: the greater one’s perceptual capacity, the more distraction irrelevant information presents, and the better the ability is to monitor the environment during task performance. An auditory version of the Attention Network Test [31] failed to demonstrate an orientation effect to Table 1. Correlation between TAIL measures. Table 1. Correlation between TAIL measures. Table 1. Correlation between TAIL measures. R Orientation(FL) Conflict (FL) Orientation(LF) Conflict (LF) Baseline (FL) Baseline (LF) Conflict (FL) 0.25 2 Orientation (LF) 0.06 0.27 2 Conflict (LF) 20.27 20.38 0.34 2 Baseline (FL) 20.08 0.32 20.08 20.24 2 Baseline (LF) 0.29 0.32 20.08 0.24 0.82* 2 Baseline (Control) 0.11 0.19 0.23 0.25 0.74* 0.68* *p,0.001. doi:10.1371/journal.pone.0053502.t001 PLOS ONE \| www.plosone.org 8 December 2012 \| Volume 7 \| Issue 12 \| e53502 New Attention Test Predicts Hearing Figure 6. TAIL results predicted auditory perception. Correla- tions between conflict resolution (left column) and baseline RT (right column) with Frequency Discrimination (top row), Backward Masking (middle row), and Simultaneous Masking (bottom row). A fitted regression line (red dashed) was plotted only for the significant correlations. doi:10.1371/journal.pone.0053502.g006 an earlier stage of processing. The nature of this asymmetry awaits further investigation, as we are unaware of similar reports or related discussion on this point. The advantageous effect of location constancy in the FL condition switched to disadvantageous in the Control detection condition. That is, response was slower for same-ear than for opposite-ear presentations. This phenomenon bears a close re- semblance to a widely documented attention effect, ‘‘inhibition of return’’ [35], referring to slower responses to a previously cued feature. The inhibitory orienting effects observed here matched or exceeded in magnitude those reported for sound frequency or location [36]. Involuntary Orientation The lack of an effect for frequency was consistent with Mondor et al.’s observation [36] of significant inhibition of return for location, but not for frequency, at a middle stimulus onset synchrony (450 ms; 500 ms on average in the current study). Inhibition of return has been reported to occur later for discrimination than for detection tasks [37], consistent with its absence in the FL and LF conditions. The nature of inhibition of return is still under debate [38], but evidence points to an exogenous (stimulus driven) mechanism that is dissociated from the operation of endogenous (goal driven) attention. Baseline RT Baseline RT (RT in absence of distracting or conflicting information) was intended to assess efficiency of information processing. Consistent with the RT literature [39], baseline RT for the discrimination conditions (FL and LF) was markedly longer than that for simple detection (Control). Despite this difference, baseline RT was highly correlated among all of the three conditions. This correlation conforms to our assumption that baseline RT reflects information processing, which remains largely the same across conditions due to the use of the same stimuli and procedures. The lack of correlation between baseline RT and the derived attention measures (involuntary orientation and conflict resolution) supports the Attention System view of attention and information processing as separate systems [15]. Though baseline RT is not an attention measure, and hence is rarely examined in attention research, it may provide a useful measure in separating contributions of attention from those of information processing and general response mechanisms. Figure 6. TAIL results predicted auditory perception. Correla- tions between conflict resolution (left column) and baseline RT (right column) with Frequency Discrimination (top row), Backward Masking (middle row), and Simultaneous Masking (bottom row). A fitted regression line (red dashed) was plotted only for the significant correlations. doi:10.1371/journal.pone.0053502.g006 doi:10.1371/journal.pone.0053502.g006 Relating TAIL to Attention Networks location as an irrelevant dimension, where the task was to discriminate the pitch of verbal stimuli. In the TAIL, the RT change caused by involuntary orientation to location and frequency was significant and of similar magnitude on the group level suggesting, according to the Load Theory, a significant portion of perceptual capacity being spared. The effect was uncorrelated between frequency and location, suggesting that orienting attention to each dimension involves different mechan- isms and/or different pathways. Error rate, however, revealed a notable location advantage on the group level. Erroneous judgments were reduced significantly by location constancy in the FL condition, but not by frequency constancy in the LF condition, from similar levels for cases of inconstancy (9.7% and 9.6% respectively). Thus, frequency constancy speeded the responses to, but did not improve the accuracy of location judgments, while location constancy improved both speed and accuracy for frequency judgments. Errors represent failures of decision making, while RTs reflect sensory processing as well as decision making. The location advantage in accuracy, but not in RT, thus hints at asymmetric depth of processing for frequency and location. Location information appears to be retained and used until a response is made, while frequency information loses influence at As the development of TAIL was informed by neuroanatomical attention theories, its outcomes can be speculatively mapped to known attention networks. First, according to the Attention Network view [14], involuntary orienting should be mediated by the frontoparietal orienting network. Corbetta and colleagues [40] divided the orienting network into two parts: a bilateral dorsal network and a right-dominant ventral network. The dorsal network orients attention and modulates sensory processing. The ventral network, inhibited during focused attention, responds to behavioral relevance of unattended stimuli and interrupts current focus of attention for reorientation. In the TAIL, task demand requires participants to direct attention to the target stimulus dimension and to ignore the irrelevant dimension. The in- voluntary orientation of attention to irrelevant dimensions would reflect inhibition of the ventral network, that is, the suppression of the competition of the irrelevant stimuli for attention. Second, executive control has been associated to anterior cingulate cortex and related brain structures [14]. According to the conflict- monitoring view [22], conflicts in information processing or decision making activates anterior cingulate cortex, which then recruits lateral frontal areas to resolve conflicts. Attention Contributions to Auditory Perception y p Our ultimate goal in developing the TAIL is to evaluate the contributions of attention components to auditory performance. As a first step towards this aim, we correlated the TAIL RT measures with threshold measures on six variants of auditory processing tasks. There are three alternative interpretations for the correlations obtained: 1) attention determines, at least in part, perceptual performance, as we have argued; 2) perceptual performance determines attention; or 3) attention and perceptual performance are both limited by a third factor. Alternative two seems unlikely for two reasons. First, the large differences between stimuli in the TAIL were perceptually highly salient (i.e. easy to distinguish). RT paradigms employing salient stimuli have been widely used to measure attention (e.g., [29,31,36]). Second, the measures of attention used in the TAIL were RT differences within conditions with similar stimulus attributes and hence similar bottom-up sensory processing. Any potential perceptual limits to TAIL performance should have been cancelled out. The third alternative, that attention and perception co-vary with a third factor, cannot be easily ruled out, but we are not aware of a plausible third factor that can account for the observed correlation patterns. It could be some other cognitive factor(s) operating in tandem with attention, for example, working memory where executive attention has been hypothesized to play an important role [43]. Disentangling attention components from related cognitive functions is beyond the scope of the current study. A co-varying factor may be neither perceptual nor cognitive, but some other individual variation independent of specific tasks, such as tiredness or anxiety level. However, in such cases, the correlation pattern would be indistinguishable for different measures of perception or attention. While bearing in mind that a list of possible co-varying factors is almost in- exhaustible, we suggest that the correlation between TAIL measures and discrimination thresholds likely reflects attention modulation of perception. We will discuss below and in detail how the results fit with the predictions of this interpretation. The stimuli used in these tasks, pure tones presented either alone or inside a noise, should pose little challenge to perceptual capacity, making involuntary attention unlikely to contribute to task performance. This was what was observed. Contributions of the two remaining TAIL measures, baseline RT and conflict resolution, indicate hierarchical and dimension-specific engage- ment of executive control in auditory performance. Relating TAIL to Attention Networks In this framework, the conflict resolution measure of the TAIL would reflect functions December 2012 \| Volume 7 \| Issue 12 \| e53502 9 PLOS ONE \| www.plosone.org New Attention Test Predicts Hearing of both the conflict monitoring and resolving networks. Another view suggests two separate networks for executive control [41]: a frontoparietal network to initiate control and a cingulo-opercular network to maintain control. In terms of this view, the conflict resolution measure of the TAIL would only capture the function of the cingulo-opercular network, because the measure was obtained during continual task performance. Examining the neural correlates of the TAIL will help illustrate the nature of the attention components measured and fit the test into our current understanding of the neural substrate of attention. It is worth noting that the attention imaging literature is primarily based on visual studies in which attention is oriented to discrete sensory events (for related auditory imaging studies, see [9,42]). The TAIL instead involves auditory stimuli and manipulation between stimulus dimensions rather than between discrete stimuli. This difference, while a cause for caution when making comparisons, could be informative to the generality of the attention models. was best illustrated in Backward Masking. When the target tone was temporally separated from the masking noise by a 50-ms silence gap (BM50, Fig. 1), perceptual thresholds were predicted by baseline RT, but not by conflict resolution, suggesting that performance was limited by the efficiency of the information processing system, with the executive control mostly inoperative. When the target tone was immediately followed by the masking noise (BM0), the difficulty of separating the task relevant (target tone) from the irrelevant (masker) information increased, and executive control was recruited. This was demonstrated by the significant correlation between BM0 threshold and conflict resolution, but not between BM0 and baseline RT. The contribution of executive control was dimension specific. The ability to focus selectively on frequency, but not the ability to focus on location, was critical. This was consistent with the nature of the stimuli, in which the target and the masker differed in their spectral but not spatial attributes. The TAIL also revealed different patterns of attention contribution across tasks. For Simultaneous Masking, performance was limited by information processing efficiency when the target tone was spectrally separated from the masker (SMN; Fig. 1). Relating TAIL to Attention Networks However, when the tone was embedded spectrally and temporally in the noise (SM), performance was not predictable from conflict resolution for either frequency or location. Considering that the spectral difference between the target-present and target-absent noise was very small, a conjecture would be that in SM attention was directed to other dimensions such as loudness. Another possibility is that Simultaneous Masking performance was limited by processes other than conflict resolution. Among the different subtypes of executive control (e.g., shifting, updating, and in- hibition [44]), conflict resolution has been modeled to result from regulation of the relative activation of competing processing pathways [45]. The regulation is triggered by conflict detection in anterior cingulate cortex and is done via sensitizing of the target pathway, inhibition of the irrelevant pathway, or a combination of the two. According to this model, for Backward Masking when the target tone is hard to separate from the noise masker (BM0), executive control can enhance performance by simply enhancing stimulus onset responses in the frequency channel from which the onset responses come and/or inhibiting sustained responses in all the other channels. For Simultaneous Masking (SMN), however, there is no clear mark of the temporal or spectral position of the target. The executive control mechanism would be inefficient in distinguishing the competing processes. This account is consistent with functional imaging evidence that anterior cingulate cortex is more active during backward than during simultaneous masking [21]. For Frequency Discrimination, the ability to focus selectively on frequency unsurprisingly predicted performance when the standard tone frequency was roved from trial to trial (FDR). When the standard frequency was fixed (FD), however, performance was predicted by neither executive control nor information processing efficiency. These observations are consistent with the proposal that performance in such circumstances depends on the formation of a memory representation of the repeated standard (’perceptual anchor’ [46]). December 2012 \| Volume 7 \| Issue 12 \| e53502 Acknowledgments We thank Cara Croft for contribution to data collection. We thank Cara Croft for contribution to data collection. References 25. Wichmann FA, Hill NJ (2001) The psychometric function: I. Fitting, sampling, and goodness of fit. Percept Psychophys 63: 1293–1313. 1. Ronnberg J, Rudner M, Foo C, Lunner T (2008) Cognition counts: a working memory system for ease of language understanding (ELU). Int J Audiol 47 Suppl 2: S99–105. 26. Brancucci A, San Martini P (1999) Laterality in the perception of temporal cues of musical timbre. Neuropsychologia 37: 1445–1451. 2. Schneider BA, Pichora-Fuller K, Daneman M (2010) Effects of senescent changes in audition. In: Gordon-Salant S, Frisina RD, Popper AN, Fay RR, editors. The Aging Auditory System. London: Springer. 167–210. 27. McKinnon MC, Schellenberg EG (1997) A left-ear advantage for forced-choice judgements of melodic contour. Can J Exp Psychol 51: 171–175. 3. Weinberger NM (2010) The cognitive auditory cortex. In: Rees A, Palmer AR, editors. The Auditory Brain. New York: Oxford University Press. 441–477. 28. Pendse SG (1977) Hemispheric asymmetry in making absolute judgments of monaurally presented pure tones. Percept Mot Skills 44: 923–928. ditors. The Auditory Brain. New York: Oxford University Press. 441–4 4. Armony JL, LeDoux JE (2010) Emotional responses to auditory stimuli. In: Rees A, Palmer AR, editors. The Auditory Brain. New York: Oxford University Press. 479–505. 29. Fan J, McCandliss BD, Sommer T, Raz A, Posner MI (2002) Testing the efficiency and independence of attentional networks. J Cogn Neurosci 14: 340– 347. 5. Moore DR, Ferguson MA, Edmondson-Jones AM, Ratib S, Riley A (2010) Nature of auditory processing disorder in children. Pediatrics 126: e382–390. 30. Morgan AL, Brandt JF (1989) An auditory Stroop effect for pitch, loudness, and time. Brain Lang 36: 592–603. 6. Humes LE (2007) The contributions of audibility and cognitive factors to the benefit provided by amplified speech to older adults. J Am Acad Audiol 18: 590– 603. 31. Roberts KL, Summerfield AQ, Hall DA (2006) Presentation modality influences behavioral measures of alerting, orienting, and executive control. J Int Neuropsychol Soc 12: 485–492. 7. Houston DM, Beer J, Bergeson TR, Chin SB, Pisoni DB, et al. (2012) The ear is connected to the brain: some new directions in the study of children with cochlear implants at Indiana University. J Am Acad Audiol 23: 446–463. 32. Simon JR, Berbaum K (1990) Effect of conflicting cues on information processing: the ’Stroop effect’ vs. the ’Simon effect’. Acta Psychol (Amst) 73: 159–170. 8. Attention Contributions to Auditory Perception The results are consistent with our prediction of increased executive control in the more challenging task variants, reflecting the greater demand for attention regulation in such conditions. The hierarchical nature To our knowledge, this is the first report of auditory performance predicted by an attention test that was administered independently of the perceptual test. The TAIL revealed differential contributions of attention control and information processing across different tasks and different levels of perceptual challenge. In this capacity, the TAIL has potential as a tool for further probing the role of attention in auditory performance. Attention control is a top-down modulating function that is particularly challenged in difficult situations. Thus, the test may be December 2012 \| Volume 7 \| Issue 12 \| e53502 December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org 10 New Attention Test Predicts Hearing extended for clinical use for separating attention from sensory contributions to impaired perceptual performance. Towards this end, our next step is to examine how TAIL measures relate to everyday auditory performance, including speech comprehension in degraded acoustic environments. Given its simplicity, the TAIL can be used with children or with older adults when presented with an appropriate interface. Children with listening difficulties are often diagnosed with auditory processing disorder, but their listening difficulty might actually result from poor attention rather than from impaired sensory processing [5,47]. Similarly, the aged population often complains of listening difficulties despite normal audiometric scores [48,49]. An independent test of auditory attention like the TAIL would help to identify how much those difficulties arise from a declining cognitive control system. speed, and of the impact of task complexity on those measures. Its use also revealed, within the scope of a limited number of auditory processing tasks, hierarchical and dimension-specific contributions of executive attention to auditory perception. Though the current study is just a first step towards specification and quantification of the contribution of attention to auditory perception, the results have demonstrated the usefulness of the TAIL in achieving this goal and potentially contributing a valid and practical clinical tool. Conclusion Conceived and designed the experiments: YXZ. Performed the experi- ments: YXZ. Analyzed the data: YXZ. Wrote the paper: YXZ JGB DRM SA. Conceived and designed the experiments: YXZ. Performed the experi- ments: YXZ. Analyzed the data: YXZ. Wrote the paper: YXZ JGB DRM SA. The TAIL provides individual and quantitative measures of multiple facets of auditory attention and information processing References p Experimental Psychology 18: 643–662. 41. Dosenbach NU, Fair DA, Cohen AL, Schlaggar BL, Petersen SE (2008) A dual- networks architecture of top-down control. Trends Cogn Sci 12: 99–105. 18. Posner MI, Snyder CR (1975) Attention and cognitive control. In: Solso RL, editor. Information Processing and Cognition: The Loyola Symposium. Hillsdale, NJ: Erlbaum. 55–85. 42. Zatorre RJ, Mondor TA, Evans AC (1999) Auditory attention to space and frequency activates similar cerebral systems. Neuroimage 10: 544–554. J 19. Amitay S, Hawkey DJ, Moore DR (2005) Auditory frequency discrimination learning is affected by stimulus variability. Percept Psychophys 67: 691–698. 43. Baddeley A (2003) Working memory: looking back and looking forward. Nat Rev Neurosci 4: 829–839. 20. Rinne T, Koistinen S, Salonen O, Alho K (2009) Task-dependent activations of human auditory cortex during pitch discrimination and pitch memory tasks. J Neurosci 29: 13338–13343. 44. Miyake A, Friedman NP, Emerson MJ, Witzki AH, Howerter A, et al. (2000) The unity and diversity of executive functions and their contributions to complex ‘‘Frontal Lobe’’ tasks: a latent variable analysis. Cogn Psychol 41: 49–100. ‘‘Frontal Lobe’’ tasks: a latent variable analysis. Cogn Psychol 4 21. van Dijk P, Backes WH (2003) Brain activity during auditory backward and simultaneous masking tasks. Hear Res 181: 8–14. 45. Cohen JD, Aston-Jones G, Gilzenrat MS (2004) A systems-level perspective on attention and cognitive control: Guided activation, adaptive gating, conflict monitoring, and exploitation versus exploration. In: Posner MI, editor. Cognitive Neuroscience of Attention. New York: The Guilford Press. 71–90. 22. Botvinick M, Braver TS, Yeung N, Ullsperger M, Carter CS, et al. (2004) Conflict monitoring: computational and empirical studies. In: Posner MI, editor. Cognitive Neuroscience of Attention. New York: The Cuilford Press. 46. Ahissar M, Lubin Y, Putter-Katz H, Banai K (2006) Dyslexia and the failure to form a perceptual anchor. Nat Neurosci 9: 1558–1564. Cognitive Neuroscience of Attention. New York: The Cuilford P 23. Moore BCJ (2003) An Introduction to the Psychology of Hearing. San Diego: Academic Press. 47. Moore DR, Rosen S, Bamiou D-E, Campbell NG, Sirimanna T (2012) Evolving concepts of developmental auditory processing disorder (APD): A British Society of Audiology APD Special Interest Group ’white paper’. Int J Audiol. In press. 24. Barry JG, Ferguson MA, Moore DR (2010) Making sense of listening: the IMAP test battery. J Vis Exp. References Cherry EC (1953) Some experiments upon the recognition of speech with one and two ears. J Acoust Soc Am 25: 975–979. 33. Botvinick MM, Braver TS, Barch DM, Carter CS, Cohen JD (2001) Conflict monitoring and cognitive control. Psychol Rev 108: 624–652. 9. Petkov CI, Kang X, Alho K, Bertrand O, Yund EW, et al. (2004) Attentional modulation of human auditory cortex. Nat Neurosci 7: 658–663. 34. MacLeod CM, MacDonald PA (2000) Interdimensional interference in the Stroop effect: uncovering the cognitive and neural anatomy of attention. Trends Cogn Sci 4: 383–391. 10. Woldorff MG, Gallen CC, Hampson SA, Hillyard SA, Pantev C, et al. (1993) Modulation of early sensory processing in human auditory cortex during auditory selective attention. Proc Natl Acad Sci U S A 90: 8722–8726. g 35. Posner MI, Rafal RD, Choate LS, Vaughan J (1985) Inhibition of Return - Neural Basis and Function. Cognitive Neuropsychology 2: 211–228. 11. James W (1890) The Principles of Psychology. New York: Holt. 36. Mondor TA, Breau LM, Milliken B (1998) Inhibitory processes in auditory selective attention: evidence of location-based and frequency-based inhibition of return. Percept Psychophys 60: 296–302. 12. Posner MI, editor (2004) Cognitive Neuroscience of Attention. New York: The Guilford Press. 466 p. 13. Cherry EC (1952) The communication of information (an historical review). American Scientist 40: 640–664. 37. Lupianez J, Milan EG, Tornay FJ, Madrid E, Tudela P (1997) Does IOR occur in discrimination tasks? Yes, it does, but later. Percept Psychophys 59: 1241– 1254. 14. Petersen SE, Posner MI (2012) The attention system of the human brain: 20 years after. Annu Rev Neurosci 35: 73–89. 38. Martin-Arevalo E, Kingstone A, Lupianez J (2012) Is ‘‘Inhibition of Return’’ due to the inhibition of the return of attention? Q J Exp Psychol (Hove). y 15. Posner MI, Petersen SE (1990) The attention system of the human brain. Annu Rev Neurosci 13: 25–42. 39. Vernon PA (1987) Speed of Information-procesing and Intelligence. New Jersey: Ablex Publishing Corporation. 16. Lavie N, Hirst A, de Fockert JW, Viding E (2004) Load theory of selective attention and cognitive control. J Exp Psychol Gen 133: 339–354. 40. Corbetta M, Patel G, Shulman GL (2008) The reorienting system of the human brain: from environment to theory of mind. Neuron 58: 306–324. g J p y 17. Stroop JR (1935) Studies of interference in serial verbal reactions. Journal of Experimental Psychology 18: 643–662. 48. Leigh-Paffenroth ED, Elangovan S (2011) Temporal processing in low- frequency channels: effects of age and hearing loss in middle-aged listeners. Journal of the American Academy of Audiology 22: 393–404. p j gy pp 50. Masson ME (2003) Using confidence intervals for graphically based data interpretation. Can J Exp Psychol 57: 203–220. 49. Pichora-Fuller MK, Souza PE (2003) Effects of aging on auditory processing of speech. International journal of audiology 42 Suppl 2: 2S11–16. 49. Pichora-Fuller MK, Souza PE (2003) Effects of aging on auditory processing of speech. International journal of audiology 42 Suppl 2: 2S11–16. 50. Masson ME (2003) Using confidence intervals for graphically based data interpretation. Can J Exp Psychol 57: 203–220. References 11 December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org December 2012 \| Volume 7 \| Issue 12 \| e53502 December 2012 \| Volume 7 \| Issue 12 \| e53502 New Attention Test Predicts Hearing New Attention Test Predicts Hearing PLOS ONE \| www.plosone.org December 2012 \| Volume 7 \| Issue 12 \| e53502 PLOS ONE \| www.plosone.org 12
https://openalex.org/W3119946285	http://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0040-1722219.pdf	English	null	Another Critical Look at Three-Phase Catalysis	Pharmaceutical fronts	2,020	cc-by	9,693	Abstract Three-phase catalysis, for example, hydrogenation, is a special branch of chemical reactions involving a hydrogen reactant (gas) and a solvent (liquid) in the presence of a metal porous catalyst (solid) to produce a liquid product. Currently, many reactors are being used for three-phase catalysis from packed bed to slurry vessel; the uniqueness for this type of reaction in countless processes is the requirement of transferring gas into liquid, as yet there is not a uniﬁed system of quantifying and comparing reactor performances. This article reviews current methodologies in carrying out such heterogeneous catalysis in different reactors and focuses on how to enhance reactor performance from gas transfer perspectives. This article also suggests that the mass transfer rate over energy dissipation may represent a fairer method for comparison of reactor performance accounting for different types/designs of reactors and catalyst structures as well as operating conditions. Keywords ►phase catalysis ►reactor performances ►mass transfer coefﬁcient ►energy dissipation rate ►energy dissipation rate internal catalytic surface; (3) adsorption of the gas and organic species onto the inner surfaces of the catalyst; (4) reaction on the inner surfaces of the catalyst; (5) desorption of the products from the surfaces; (6) diffusion of the products from the interior through the pores to the external surfaces; (7) mass transfer of the products from the external surfaces to the bulk ﬂuid. THIEME Review Article e117 Another Critical Look at Three-Phase Catalysis Xiong-Wei Ni1 Xiong-Wei Ni1 Address for correspondence Xiong-Wei Ni, PhD, School of Engineering and Physical Sciences, Heriot–Watt University, Edinburgh EH14 4AS, United Kingdom (e-mail: x.ni@hw.ac.uk). 1School of Engineering and Physical Sciences, Division of Chemical Engineering, Heriot–Watt University, Edinburgh, United Kingdom Pharmaceut Fronts 2020;2:e117–e127. Pharmaceut Fronts 2020;2:e117–e127. © 2020. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany Article published online: 2020-12-31 Article published online: 2020-12-31 Article published online: 2020-12-31 Article published online: 2020-12-31 THIEME Introduction P (bar) g H2 dissolved/g H2O mol H2 dissolved/mol H2O 1 1.54 106 1.39 105 2 3.09 106 2.78 105 3 4.62 106 4.16 105 5 7.72 106 6.94 105 reactions. In order for any reactive gas to arrive at the surfaces of catalysts that are surrounded by liquid, macro- scopic bubbles must be broken into smaller and minute ones, the latter would reach equilibrium with liquid and become dissolved at the microscopic/nanoscopic level, and it is thus the dissolved gas in liquid, not visible bubbles, that holds the key for mass transfers in multiphase catalysis. From the chemical engineering viewpoint, how to increase the dissolved gas specious in liquid is an effective measure of how efﬁcient various reactors are for carrying out heterogeneous catalyses. Fig. 1 Reaction mechanism of hydrogenation involving spherical solid catalyst particles. as illustrated in ►Fig. 2. Since the reaction rate is proportional to CH2surface, three-phase catalysis is often severely restricted by the limitations in the aforementioned mass transfers, affecting the reaction rate, selectivity, productivity, and pro- longing reaction times.7 From chemical engineering perspec- tives, how to minimize the difference of (CH2Bulk CH2surface) is the key in ensuring effective heterogeneous catalysis. Note that improving the structure of catalysts, e.g., mono- liths, would enhance the areas of catalyst surfaces, and the subsequent reaction efﬁciency.10–12 Reactors with monolith catalyst packing are hydrodynamically superior to existing industrial reactors13; however, the physical transport process of gas dissolution into liquid must be driven by ﬂuid mechanic forces, in combination with reactor designs/additions and catalyst structure. In 1924, Murray Raney, an American engineer, discovered that by fusion of a 50:50 Ni/Al alloy and then leaching out the Al using aqueous NaOH, a nickel sponge was obtained, which was much more active than other commercial catalysts.8,9 The Raney nickel catalysts are often large in diameter, e.g., 1 to 10 mm, it is easier to imagine mass transfer of gas species into these catalyst spheres. For modern metal catalysts, their sizes are usually range between micro and nanometers, thus, it becomes more difﬁcult to envisage the aforementioned mass transfer processes taking place physically within these minute catalyst particles, but the truth is that all occur at the molecular level. Introduction Three-phase catalysis is one of the key synthesis steps in petrochemical, chemical, cosmetic, pharmaceutical, and food industries, for instance, converting alkenes and aromatics to saturatedalkanes(parafﬁns)and cycloalkanes (naphthenes)in petrochemical industry to reduce toxicity and reactiveness; converting unsaturated alkenes, alkynes, aldehydes, imines, and nitriles to alcohols and amines in chemical/pharmaceuti- cal industries to produce purer products; processing vegetable oils into solidor semisolid fats,e.g., margarine, infoodindustry to add values, aid transportation, and lengthen products shelf times. To initiate any three-phase catalysis, e.g., hydrogena- tion,itrequiresbothgas(H2)andliquidreactantsdiffusinginto the surfaces of solid catalysts, as illustrated in ►Fig. 1 focusing on one single spherical catalyst particle of a radius R. The reaction mechanisms of many commercial heterogeneous catalyses, although successfully operated, are still a matter of debate and controversy, and generally consist of seven well- knownsteps1–4:(1)masstransferofgas,e.g.,H2, frombulk into the liquid phase and then to the external surface of a catalyst particle; (2)diffusionofboththedissolved H2 (gas) andorganic (liquid) compounds through the pores of the catalyst to the The steps (3) to (5) are regarded as the catalytic reaction, while the rest of the steps are associated with mass transfer. These reaction and transport processes occur concurrently in such catalyses.5,6 The degree of resistance in mass transfer increases signiﬁcantly from liquid–solid systems to gas–solid and further to gas–liquid–solid catalysis. For liquid–solid catalysis, solid catalyst particles are readily wetted by the surrounding liquid, the dispersion of liquid into the pores of the catalyst is a relatively straightforward process. For three- phase catalysis, on the other hand, the gas reactant must “travel” through the gas–gas interface, gas–liquid interface, and then the gas–solid interface before reaching the outer surfaces of the catalyst. In each of the boundary crossings, the concentration of gas is reduced, leading to the arriving con- centration of H2 at the surfaces of solid catalysts (CH2surface) being signiﬁcantly smaller than that of the input gas (CH2Bulk), received October 23, 2020 accepted December 3, 2020 © 2020. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany DOI https://doi.org/ 10.1055/s-0040-1722219. ISSN 2628-5088. Another Critical Look at Three-Phase Catalysis Ni e118 Table 1 Dissolved hydrogen in water at 298 K14 Fig. 1 Reaction mechanism of hydrogenation involving spherical solid catalyst particles. Trickle Bed Reactors Trickle Bed Reactors A trickle bed reactor is a variant of packed bed where the liquid solvent is showered down from the top, thus increasing the surface area of the liquid, and gas can go either co-currently or counter-currently with the liquid. Hydrodynamics of trickle bed reactors were studied15,16 using transport modeling,17 computational ﬂuid dynamics (CFD) modeling,18 electrical resistance tomography,19 as well as by high pressures.20 Due to the reliability of their operation, trickle bed reactors have won a great use in oil industry, and also found applications in SO2 oxidation,21 glucose hydrogenation over ruthenium cata- lyst,22 hydro-treating atmospheric residue,23 hydro-puriﬁca- tion,24 catalytic hydro-treatment of vegetable oils,25 fuel production via Fischer–Tropsch synthesis,26 hydrogen produc- tion by aqueous-phase reforming of xylitol,27 hydrogenoly- sis,28,29 continuous thermal oxidation of alkenes with nitrous oxide,30 liquid-phase selective hydrogenation of methylacety- lene and propadiene,31 hydrogen peroxide,32 as well as contin- uous operation.33 Reactors for Three-Phase Catalysis Mixingand reactordesignsplayacriticalroleinheterogeneous catalysis. The commonly used reactors in industrial multi- phase catalyses are generally divided into two categories: suspended or ﬁxed bed reactors. The former is associated with mobile catalyst particles that are suspended in reactors, also referred as slurry reactors, including bubble columns (suitable only for very small catalyst particles), agitated tanks, and three-phase ﬂuidized beds. The ﬁxed bed reactors involve stationary catalysts, including packed bed, trickle bed, and pulsed bed reactors. ►Fig. 3 illustrates the basic principles of the two types of reactor set-ups. In the following sections, each type of the reactors is assessed in terms of the common yardsticks for transferring gas into liquid, such as (1) mechanisms of breaking and maintaining gas bubble sizes; (2) mass transfer coefﬁcient (kLa) per energy dissipation rate (W m3), as mass transfer coefﬁcient alone cannot provide any meaningful comparison of reactor performances; and (3) where the dissolved gas concentration is measured in each type of contactors is critical, as it differentiates local from overall mass transfer rate. Therearetwopossiblemechanismsintricklebedreactorsfor breakingdownbubblesizesandinitiatingdissolutionofgasinto liquid: (1) the interactions of liquid and gas ﬂows, (2) through the tortoise routes that are formed by the packed catalysts, the denser the solid particles, the smaller the diameters of bubbles soformed.Therearehowevernofacilitiesofmaintainingbubble sizes in such reactors, and once gas has passed the dissolution zones, bubble coalescence occurs readily. The determinations of gas–liquid or liquid–solid mass transfer coefﬁcients in packed beds were performed in systems involving either nonreaction schemes, for example, absorption/desorption of O2 or CO2 in water,34–37 or actual reactions.38,39 Iliuta et al compiled mass transfer data from more than 3,200 experiments in 52 gas– liquid systems, with over 60 packing sizes/geometries and 17 column diameters40; however, there were neither information on where dissolved gas concentration was measured nor on energy dissipation rates in trickle bed reactors. Packed Bed Reactors In a packed bed reactor (PBR), stationary solid catalyst particles are either packed or shelved in the reactor. The main choice for design and operation with this type of reactors is the direction of ﬂow for both gas and liquid phases, e.g., co- or countercurrent. Fig. 3 Reactor type for three-phase catalysis: slurry bed (left) and packed bed (right). Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Introduction Visible bubbles with a diameter from 100s to 1000s micrometers are at the macroscopic level, similar to catalyst particles, mixing, catalyst surface structure, and physical interactions that affect the outcome of catalytic Majority of three-phase catalysis is operated at elevated pressures,aspressureincreases thesolubilityofgasintoliquid. ►Table 1 displays such an effect where four times more hydrogen is dissolved in water at 5 bar compared with that at 1 bar.14 However, it should be noted that the amount of hydrogen dissolved in water is measured in terms of micro- grams per gram of water, i.e., parts per million. As a result, the effect of increasing pressure as a means of increasing Fig. 2 Mass transfer through phase boundaries in three-phase catalysis. Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Fig. 2 Mass transfer through phase boundaries in three-phase catalysis. Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Another Critical Look at Three-Phase Catalysis Ni e119 CH2surface is rather small in three-phase catalysis; subse- quently how to improve mass transfer of gas into liquid is THEfundamental chemical engineering parameter in most, if not all, of heterogeneous catalyses in industries. This is the focus of this review article. Stirred Tank Reactors Stirred tank reactors (STRs) are the widely used industrial workhorse for chemical reactions, including heterogeneous catalysis,55–57 catalytic cracking,58 catalytic foaming,59 and Fischer–Tropsch synthesis,60 just to name but a few. In terms of operation, solvent and catalyst particles are added to the tank, and impeller or impellers are used for mixing and suspending mobile catalyst solids. A sparger is implemented for introducing gas, either from the top or the bottom, with or without recycling of the product stream back to the reactor. Jet ﬂow/loop reactors are the variants of bubble columns; the implementation of jet is to break gas bubbles and the looping is to increase the time for dissolution of gas. In jet loop reactors, an external pump is used to circulate liquid (along with the catalyst and often some gas) through an ejector type nozzle, as such gas-inducing nozzle is a critical design compo- nent of jet loop reactors,111,112 Fundamental scientiﬁc studies of ﬂow in and out of a nozzle were reported in terms of mixing,113,114 hydrodynamics,115–117 bubble size distribu- tion,118 mass transfer,119,120 as well as reaction kinetics.121 The mechanisms of breaking gas bubbles in STR usually include the rotating motions of impeller(s) and the inter- actions of ﬂuid with the presence of wall bafﬂes. In labora- tory-scale STRs, mixing is regarded as uniform, thus, the dissolved gas concentration measured anywhere in the reactor can be used for the determination of the overall mass transfer coefﬁcient. With the increase of reactor vol- ume, however, a gradient in bubble sizes is often generated, in which the smaller bubbles appear near the tip of the impellor, and the larger bubbles appear elsewhere. The balance between bubble breaking and coalescence is deter- mined by the hydrodynamic force of impeller rotation in the presence of mixing aids, e.g., wall bafﬂes, thus the designs of impeller and turbulence promoter61 are critical in providing more uniform bubble size, and in turn the higher mass transfer rate,62,63 e.g., mass transfer using either a dual impeller64 or radial–axial impeller combination65 was 15 to 35% higher in comparison to a single impeller. Slurry Bed Reactors In slurry bed reactors, solid catalyst particles are suspended in a liquid solution. There are many means of creating slurry suspension, e.g., mechanic, pneumatic, oscillatory, and com- binations of ﬂow directions of gas and liquid phases. Pulsed Trickle Bed Reactor In pulsed trickle bed reactors (a variant of packed bed), either gas or liquid ﬂow is subjected to a pulse motion41 that can be generated using hydropneumatic, self-propelled, or elastic membranes.42 The pulses cause the transition to bubble ﬂow, while the parts of the bed in between pulses reside at the transition to trickle ﬂow. Properties of pulsing ﬂow were studied,43–45 including hydrodynamics of trickling to pulsing ﬂow transition46 and bubbly to pulsing ﬂow regimes,47–49since the majority of industrial processes operate at or near the transitionfromtrickling topulsing ﬂow.Theoperationoftrickle bed reactors at elevated temperatures was also reported.50 bed reactors at elevated temperatures was also reported. The same mechanisms for breaking bubbles and initiating dissolution of gas into liquid in trickle bed reactors apply to pulsed trickle beds, with an additional feature of pulsing. The purpose of pulsing liquid is effectively to “hold” bubbles for a fraction of time (e.g., 0.5 Hz), this enhances overall heat and mass transport while reducing axial dispersion. Keeping all other parameters constant, reactor operation in the presence of pulses resulted in up to 30% increase in reaction rate,44 15% increase in styrene concentration,51 and 45% improvement in Fig. 3 Reactor type for three-phase catalysis: slurry bed (left) and packed bed (right). Another Critical Look at Three-Phase Catalysis Ni 120 e120 styrene selectivity in hydrogenation of phenylacetylene over Pt/γ-Al2O3 catalyst compared with that without. Some mass transfer data in pulsed tricklebed reactors were reported,52–54 once again, no information was given on both where dissolved gas concentration was measured and energy dissipation rate. the stirrer (s1), Lh and ρ are the height (m) and density (kg m3) of the liquid in the reactor, and Ds and Dv the diameters of the agitator and the vessel (m), respectively. This is the energy that is received and utilized by the reaction media to achieve the measured mass transfer rate in the said reactor.87–94 Available mass transfer and energy dissipation data are used for comparison in a later section. Bubble Column A bubble column by deﬁnition consists of a column ﬁlled with liquid and solid catalyst particles with gas being sparged from the bottom. The hydrodynamics and heat/mass transfer of slurry bubble columns were reported.68,95–106 In addition, slurry bubble columns were also used in Fischer–Tropsch synthesis,107,108 CO methanation over La-promoted Ni/Al2O3 catalyst109 and green fuel production via hydrocracking of vegetable oil.110 It should however be noted that the bubble column itself has no physical mechanisms of breaking bubbles as well as preventing bubbles from coalescing, and various restrictions, delays, and recycles are implemented to aid the physical process of bubble breaking, e.g., down pipes, loop, jet, and pulsing. Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Stirred Tank Reactors Mass transfer of a gas into a liquid has intensively been studied in STR, such as air in water,66–68 H2 in water,69–72 O2 in liquid hydrocarbons,73 and in n-octacosane processes,74 in fermen- tation vessels,75 in gas–liquid–solid systems,76–78 and in scale-up STRs.79,80 Modeling of gas–liquid mass transfer81 was performed using an Euler–Lagrange approach82 and CFD.83 Additionally, liquid–solid mass transfer84,85 and sol- id–liquid mass transfer86 were also reported in STR. The jet in jet loop reactor sends liquid plume downwards, causing dispersion and entrainment of minute bubbles, this is wherethe maximum dissolutionofgas into liquidand excellent gas–liquidmasstransfer takeplace.Whenthejet plumereaches the bottom of the down-ﬂow, the jet stream comes up, leading to bubble coalescence and visible bubbles rising. Effectively the dissolution zone is the length of the jet core. A study by Mandal114 shows that the core length was 30 to 45mm in a 1,500-mm tall column of a diameter of 52mm, i.e., approxi- mately 3% of the full length, although the core length can reach 50% of the full reactor length in industrial-scale operations. Dissolved gas concentrations were mainly measured at the tail end of the core, where bubble sizes were generally from 1 to 6mm.122,123 The energy requirement for forming such a jet is usually high, e.g., 1.2 to1.5 m3/h forliquidﬂowand 0.25 to1 m3/ h for gas ﬂow in a jet loop reactor of 200mm diameter and 700mmtall118;however,neitherequationsnordataweregiven on energy dissipation rates due to the combination of wide- ranging ﬂuid mechanical zones with nonstandardized designs of jet promoters for this type of reactors. Energy dissipation rates in STR takes the form of Equation 1: Jet loop reactors have been used for model reactions such as hydrogenation, chlorination, phosgenation, hydroformyla- tion,124,125 as well as for processes of CO2 absorption,126 anaer- obic codigestion of olive mill wastewater and liquid poultry manure,127 treatment of slaughterhouse wastewater,128 imida- cloprid preparation,129 and microbial fermentation.130 Where P/V is the energy dissipation rate per volume (W m3), P0 the power number for the impeller depending on the impeller type and dimensions, N the rotational speed of Another Critical Look at Three-Phase Catalysis Ni e121 intensity of mixing in a batch OBR can be controlled by varying either oscillation amplitude or/and frequency when the oriﬁce diameter and bafﬂe spacing are ﬁxed. Fluidized-Bed Catalytic Reactors Heterogeneous catalysis has also been performed in ﬂuid- ized-bed reactors where the gas reactant is bubbling through a liquid bed containing a solid catalyst.131 The desirable features of a ﬂuidized bed reactor can brieﬂy be summarized as its the favorable heat transfer, temperature uniformity, high effectiveness factors, low pressure drop, and ability to add/remove catalysts, and some of the disadvantages are entrainment, attrition, wear, as well as nonuniform resi- dence time distributions, and unpredictability.131 In reality, the design and scale-up of ﬂuidized-bed reactors rely heavily on experience, mechanistic understanding, and models.132 There is a void in the literature for the evaluation of energy dissipation rate in ﬂuidized bed reactors. Where Nb is the number of bafﬂes per unit length in OBR (m1),αthe ratioof the effective oriﬁceareatothetubearea,x0 the oscillation amplitude (m), ω the angular oscillation fre- quency (radians s1) and CD the oriﬁce discharge coefﬁcient. In summary, the advantages of PBRs are (1) there is lesser demand on particle size of catalyst; (2) it is relatively easy to design this type of reactors; and (3) there is no need for catalyst separation after reaction, reducing unit operation and associated requirement for energy and labor. The major drawback for stationary catalysts is that the required quan- tity of the catalyst is signiﬁcantly higher in comparison to mobile catalyst arrangement for the same conversion due to reduced surface areas of the catalysts. In addition, bubble sizes are relatively large, as there is generally lack of means of holding small bubbles, leading to lower mass transfer capa- bility and longer reaction times. When gas is involved, gas bubbles are broken down by the formed vortices; the reciprocal movement of the bafﬂes also holds and maintains bubble sizes. When compared with mass transfer of air in water,149–151 air in cultures,152 O2 in water,153,154 ozone in water,155,156 and CO2 in water157 in STRs or bubble columns, kLa values in OBR were much improved, which was attributed to three distinct features: (1) smaller and more uniform bubble sizes due to the mechanism of maintaining bubble sizes; (2) higher gas hold-up, as a foaming layer at the top of the column acts as a “blanket” to prevent minute bubbles from disengaging, and the oscillatory motion drags these bubbles back into the liquid; and (3) signiﬁcantly prolonged residence times of bubbles due to the reciprocal motion of eddies. Micro Bubbles Process intensiﬁcation in multiphase reactors have been used133–135 with various mechanisms, e.g., continuous ﬂow mixing vessels,136 vorticial ciliary ﬂows,137 micro- mesh,138 microbubble generators,139 microreactors,140–143 and in scale-up ﬂow reactors.144 The key feature of these intensiﬁed devices is the ability of producing ﬁne and minute bubbles; consistently, some show the capability of maintain- ing ﬁne bubble sizes, leading to enhanced mass transfer rates. However, currently, there are no publications on energy dissipation rates in these new/novel reactors. Under the same reaction conditions, rising proﬁles of the initial reaction rate against energy dissipation are still seen for all pressures tested in the OBR (►Fig. 5), indicating that the capacity of mixing is not only signiﬁcantly larger, but also more energy efﬁcient than the PARR reactor, for example, approximately six times less energy dissipation in the OBR was required to achieve the same reaction rate obtained in the PARR working at the same pressure or approximately three times less energy dissipation if the operating pressure in the OBR was halved. Fluidized-Bed Catalytic Reactors For slurry bed reactors, there are mechanic means of breaking bubbles and achieving gas dissolution, thus increas- ing mass transfer and reaction rates; mobile catalysts offer signiﬁcantly higher surface areas, leading to much less catalyst and shorter reaction time to achieve the same conversion in comparison to stationary catalysts. The shortcoming for this type of operation is that a separation of solid catalyst particles is mandatory at the end of reaction, leading to potential loss of catalyst due to attrition. Filtered catalyst particles can be reused for a few times depending on processes. The above key features are manifested in a comparative study of catalytic hydrogenation of 3-butyn-2-ol over Pd/ Al2O3 catalyst to generate 3-buten-2-ol (an intermediate) in both a commercially available stirred tank PARR reactor (PARR in short) and an OBR,158 where an increase in the initial reaction rate (r0) with the increase of energy dissipa- tion rate (stirring speed) is seen up to 29,500 W m3 in the PARR reactor (►Fig. 4), beyond which r0 is unaffected, indicating that the capacity of mixing in terms of stirring speed in the PARR has reached its ceiling and no longer affects the reaction rate. Oscillatory Bafﬂed Reactors Oscillatory bafﬂed reactors (OBR) generally consist of a jacketed cylindrical column and a set of oriﬁce bafﬂes. The up and down movement of the bafﬂe set within the column generates intensive eddy current that moves ﬂuids from wall to center, creating equal radial and axial velocity compo- nents, which is the essential measure of uniform mixing. The Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Stirred Tank Reactors Oscillatory amplitudes from half to one bafﬂed cell length and oscilla- tion frequency from 1 to 8 Hz can be employed, the latter is much higher than that in pulsed trickle bed reactors. The energy dissipation rate in OBR was developed by assessing inertial and frictional effects of the ﬂow together with pressure drop due to a static head,145–148 as in Equation 2: Comparison In this article, several reactors for heterogeneous catalysis have been introduced, each involves different physical Another Critical Look at Three-Phase Catalysis Ni e122 Fig. 4 Effect of energy dissipation (P/V) on initial reactor rate (r0) in the stirred tank PARR reactor at 1 bar.148 transfer coefﬁcient or bubble mean size alone does not serve any meaningful purpose of comparison, since higher energy dissipation experienced by the reaction mixture generally leads to higher mass transfer rates. By dividing mass transfer coefﬁcient over energy dissipation that is consumed by reaction media to generate the measured transfer rate would offer a fairer and better comparison of reactor performances, counting for different designs and operating conditions. This ratio is used in this article. While there are plentiful research papers on the evaluation of energy dissipation in both STRs and OBRs, no research articles are found for other types of both slurry and packed bed contactors. In some cases, data of mass transfer coefﬁcient or mean bubble sizes or gas hold-up were presented as a function of energy dissipation, but no information was given on how these dissipation rates were derived. Taking a leap of faith, ►Table 2 compiles the ratio of kLa over their correspond- ing energy dissipations as thekey indicator (last column). Note that the lowest energy dissipation data were taken for com- parison in ►Table 2, and this is due to the unavoidable fact in gas–liquid systems where the percentage increase in mass transfer coefﬁcient is much smaller than that in energy dissipation (mixing), e.g., often 100% increase in energy dissi- pation leads to 1 to 5% increase in mass transfer rates. Fig. 4 Effect of energy dissipation (P/V) on initial reactor rate (r0) in the stirred tank PARR reactor at 1 bar.148 Fig. 4 Effect of energy dissipation (P/V) on initial reactor rate (r0) in the stirred tank PARR reactor at 1 bar.148 Fig. 5 Effect of energy density (P/V) on initial reaction rate (r0) at different pressures in PARR and OBR. Working conditions: initial molar ratio 3-butyn-2-ol/Pd ¼ 1,360 and temperature ¼ 323 K.148 OBR, os- cillatory bafﬂed reactor. Comparison While the ratio of kLa over (P/V) in ►Table 2 gives the indicative comparison of the capability of delivering gas to liquid mass transfer for different types of gas–liquid con- tactors, there are three factors to note: • Each reactor type has means of breaking bubbles; it is however the mechanism of maintaining achieved bubble sizes that are critical to the overall mass transfer rate, as gas bubbles coalesce naturally. Reactors with higher ratios of mass transfer over energy dissipation are generally equipped with better mechanisms of maintaining bubble sizes. Fig. 5 Effect of energy density (P/V) on initial reaction rate (r0) at different pressures in PARR and OBR. Working conditions: initial molar ratio 3-butyn-2-ol/Pd ¼ 1,360 and temperature ¼ 323 K.148 OBR, os- cillatory bafﬂed reactor. Fig. 5 Effect of energy density (P/V) on initial reaction rate (r0) at different pressures in PARR and OBR. Working conditions: initial molar ratio 3-butyn-2-ol/Pd ¼ 1,360 and temperature ¼ 323 K.148 OBR, os- cillatory bafﬂed reactor. • Most gas–liquid contactors exhibit nonuniform mixing patterns, or have different ﬂuid dynamic zones, where exactly the dissolved gas concentration was measured in a given reactor can have signiﬁcant impact on the determi- nation of the overall mass transfer rate; unfortunately very few details in this aspect were disclosed in published papers. designs and operating conditions. How could reactor per- formances be compared and what would be the common basis for such a comparison? Using the 100 m sprint as an example, if you race against Usain Bolt, Usain Bolt will win every time, because Usain Bolt is the fastest sprinter in the world, while you are an amateur runner. We accept the outcome without questioning. If you challenge Usain Bolt with you on a motor bike, you will win the race. This leads to the key question: what is the common basis for comparison? From the above example, power consumed by the motor bike is more than that by the human being, and the sprint time divided by the power consumption would provide a level- playing ﬁeld for comparison of different racing modes; in this case Usain Bolt will win it again. The exact principles are applied here. Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Comparison The basis is the energy dissipation rate (W m3) that is received by the reaction media in a given reactor, which is neither the electric power of the motor that is used for stirring in STRs nor the power of pump that is employed for generating jets in jet loop reactors. Using either mass designs and operating conditions. How could reactor per- formances be compared and what would be the common basis for such a comparison? Using the 100 m sprint as an example, if you race against Usain Bolt, Usain Bolt will win every time, because Usain Bolt is the fastest sprinter in the world, while you are an amateur runner. We accept the outcome without questioning. If you challenge Usain Bolt with you on a motor bike, you will win the race. This leads to the key question: what is the common basis for comparison? From the above example, power consumed by the motor bike is more than that by the human being, and the sprint time divided by the power consumption would provide a level- playing ﬁeld for comparison of different racing modes; in this case Usain Bolt will win it again. The exact principles are applied here. The basis is the energy dissipation rate (W m3) that is received by the reaction media in a given reactor, which is neither the electric power of the motor that is used for stirring in STRs nor the power of pump that is employed for generating jets in jet loop reactors. Using either mass • The contribution of static pressure head, e.g., ρgug, or ρLgHsQg, or ρgvgVL in ►Table 2, to the overall energy dissipation is generally very small; the inclusion of such a term makes little difference in energy dissipation data compared with the exclusion of it. 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Comparison In this article, the mass transfer rate over energy dissipation is proposed as a fairer method for comparison of Another Critical Look at Three-Phase Catalysis Ni e123 Table 2 Comparison of mass transfer rates based on energy dissipation Reference Reactor type Energy dissipation (P/V) equation P/V (kW m3) kLa (s1) Ratio kLa/(P/V) 159 Sparged stirred tank P/V ¼ (P0f3D5ρN þ ρgvgVL)/VL P0–power number f – agitation speed N – number of impellor D – diameter of impellor vg – gas superﬁcial velocity VL – volume of liquid 0.1 0.02 0.2 160 Sparged stirred tanks P/V ¼ α(P0 2ND3/Q0.56)β/VL N – agitation speed D – stirrer diameter Q – gas ﬂow rate P0–energy input in unaerated system α and β depend on type and numbers of stirrer 0.15 0.002 0.133 89 Sparged stirred tanks P/V ¼ (2πNM þ ρLgHsQg)/VL N – agitation speed M – torque Hs – liquid height Qg – gas ﬂow rate VL – liquid volume 1.3 0.04 0.03 161 Sparged stirred tank P/V ¼ 2πNM/VL N – agitation speed M – torque VL – liquid volume 0.7 0.019 0.027 162 Jet ﬂow loop 0.8 0.014 0.018 120 Jet loop 2 0.6 0.3 163 Plunging jet bubble column 8 0.2 0.025 111 Gas–liquid ejector 20 1.1 0.055 164 Microbubble nozzle 0.05 0.002 0.04 Perforated plate 0.005 0.0002 0.04 Spiral liquid ﬂow 1.3 0.001 0.008 Venturi 1 0.0003 0.003 Ejector 7 0.0004 0.00006 Pressurized dissolution 10 0.001 0.0001 138 Jet array downﬂow bubble column 1.254 0.139 0.111 149 Oscillatory bafﬂed column (P/V) ¼ 2ρNb(1 α2)x0 3ω3/(3πCD 2α2) Nb – number of bafﬂes per unit length CD – oriﬁce discharge coefﬁcient α – bafﬂe-free cross-sectional area x0–oscillation amplitude ω – angular frequency of oscillation 0.05 0.02 0.4 153 Oscillatory bafﬂed column (P/V)0 ¼ 2ρNb(1 α2)x0 3ω3/(3πCD 2α2) Nb – number of bafﬂes per unit length CD – oriﬁce discharge coefﬁcient α – bafﬂe-free cross-sectional area x0–oscillation amplitude ω – angular frequency of oscillation (P/V)B ¼ ρgug P/V ¼ (P/V)0 þ (P/V)B 0.02 0.005 0.25 Table 2 Comparison of mass transfer rates based on energy dissipation Pharmaceutical Fronts Vol. 2 No. 3/2020 © 2020. The Author(s). Another Critical Look at Three-Phase Catalysis Ni e124 reactor performance accounting for different types/designs of reactors and catalyst structures as well as operating conditions. Hope more papers on energy dissipation rates for both existing and new-type reactors are emerging to ﬁll the gap. Conﬂict of Interest Conﬂict of Interest The authors declare no conﬂict of interest. The authors declare no conﬂict of interest. 23 Alonso F, Ancheyta J, Centeno G, Marroquín G, Rayo P, Silva- Rodrigo R. Effect of reactor conﬁguration on the hydrotreating of atmospheric residue. Energy Fuels 2019;33(02):1649–1658 Funding Funding None. Funding 21 Haure PH, Hudgins RR, Silveston PL. Periodic operation of a trickle-bed reactor. AIChE J 1989;35(09):1437–1444 g None. 22 Gallezot P, Nicolaus N, Flèche G, Fuertes P, Perrard A. Glucose hydrogenation on ruthenium catalysts in a trickle-bed reactor. 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W2989386571.txt	https://link.springer.com/content/pdf/10.1007%2F978-3-030-27731-4_2.pdf	en		A High-Quality Finger Vein Dataset Collected Using a Custom-Designed Capture Device	Advances in computer vision and pattern recognition	2,019	cc-by	4,549	Chapter 2 A High-Quality Finger Vein Dataset Collected Using a Custom-Designed Capture Device Raymond Veldhuis, Luuk Spreeuwers, Bram Ton and Sjoerd Rozendal Abstract High-quality finger vein datasets available for the research community are still relatively scarce; therefore, we collected a set of finger vein images of high resolution and a known pixel density. Furthermore, this is the first dataset which contains the age, gender and handedness of the participating data subjects as metadata. This dataset has been collected using a custom-designed biometric capture device. The various aspects of designing this biometric capture device are addressed in this chapter. New insights and continuing work on the design of better capture devices have led to novel ideas which are presented in this chapter. To justify the importance of this dataset, performance figures in terms of EER of several well-established algorithms using this dataset and an existing dataset are compared side by side. Keywords Finger vein capture device · Finger vein data set · 3D finger vein reconstruction 2.1 Introduction The vascular or vein pattern of the finger is advertised as a promising new biometric characteristic. Biometric recognition based on finger vein patterns is characterised by very low error rates, good presentation attack resistance and a user convenience that is equivalent to that of fingerprint recognition. Though this new form of biometrics is already commercially deployed, it still lacks a strong scientific base. This is due to R. Veldhuis · L. Spreeuwers (B) · B. Ton Data Science Group, Faculty of EEMCS, University of Twente, Enschede, The Netherlands e-mail: l.j.spreeuwers@utwente.nl R. Veldhuis e-mail: r.n.j.veldhuis@utwente.nl B. Ton e-mail: b.t.ton@alumnus.utwente.nl S. Rozendal University of Twente, Enschede, Netherlands e-mail: s.p.rozendal@student.utwente.nl © The Author(s) 2020 A. Uhl et al. (eds.), Handbook of Vascular Biometrics, Advances in Computer Vision and Pattern Recognition, https://doi.org/10.1007/978-3-030-27731-4_2 63 64 R. Veldhuis et al. industrial protectiveness, which restricts the ability to verify claimed performances. In order to compare existing algorithms, a standardised testing method is needed and more datasets should be made available to researchers. In order to stimulate the academic research on vascular pattern recognition, this chapter will present a finger vascular pattern dataset which has recently been made available to other researchers [17]. The presented dataset is unique in its kind as it provides high-resolution images together with demographics about the data subjects. Another contribution of this chapter is the performance verification of several published algorithms using both the newly collected dataset and an existing dataset collected by the Peking University [12]. Our dataset has been collected using a custom-designed capture device. The various aspects of designing this capture device are also covered in this chapter. In the remainder of this chapter, first a brief overview is provided of finger vein acquisition techniques and systems in Sect. 2.2. Next, the custom-designed capture device is described in detail (Sect. 2.3), followed by the dataset (Sect. 2.4). In Sect. 2.5, results of various finger vein recognition algorithms on the database are presented. Section 2.6 presents the next-generation finger vein scanner currently under development at the University of Twente: a more compact design with 3D capabilities and other enhancements. Section 2.7 presents conclusions and in Sect. 2.8 future work is described. 2.2 Overview of Finger Vein Acquisition Systems 2.2.1 Types of Sensors We first briefly summarise the different types of sensors for finger vein recognition and then present our own design. Devices that capture the vascular pattern inside a finger are based on the fact that the haemoglobin inside the veins has a higher absorption of Near-Infrared Light (NIR light) than the surrounding tissue. This means that the vascular pattern inside a finger can be captured by a device that is sensitive to NIR light. The veins have to be made visible with NIR light, but there are multiple possibilities to illuminate the finger. The main types that are found in existing devices are shown in Fig. 2.1. The illumination with the light reflection method is on the same side as the camera. This allows the device to be more compact. During operation, the user of the device can still see his finger. The disadvantage of this method is that the image sensor mainly captures the reflected light from the surface of the finger, because the light shallowly penetrates the skin. Hence, this method gives images with low contrast between tissue and veins. The light transmission method does deliver high-contrast vascular pattern images, because the light passes through the finger and no reflections of the surface are captured. The illumination is at the other side of the finger relative to the camera. The disadvantage of this method is that the user has to put his finger into the device such that he cannot see his finger anymore, which can cause discomfort. The third illumination type is side lighting method. This method still allows an open 2 A High-Quality Finger Vein Dataset Collected … 65 NIR light source vein vein NIR light source camera NIR light source reflection NIR light source camera transmission vein NIR light source camera side Fig. 2.1 Reflection, transmission and side illumination acquisition device such that the user can see his finger. The light sources are placed on either one side or both sides of the finger. NIR light goes through the sides of the finger and scatters there, before it is captured by the image sensor. This method does allow for high-contrast images. However, the sides of the finger are overexposed in the images. Some examples of commercially available sensors and sensors developed by academics are presented in Sects. 2.2.2 and 2.2.3. For a more complete overview, please refer to Chap. 3 of this book. 2.2.2 Commercial Sensors There are several devices on the market for vascular pattern recognition. The market leader in finger vein capture devices is Hitachi. They have developed multiple systems that are capable of capturing finger vein images using light transmission or side illumination. Hitachi claims that it has a False Non-Match Rate (FNMR) of 0.01% at a False Match Rate (FMR) of 0.0001% [3, 4]. However, it is hard to verify these claims, because the devices and image data are not accessible. Another company that builds finger vein capture devices is Mofiria, a daughter company of Sony. This company also produces various devices among which one using light transmission, but where the finger is placed sideways on the sensor. They claim an FNMR of 0.1% at an FMR of 0.0001% [15], but again these are closed devices and data are not accessible. 2.2.3 Sensors Developed by Academics At several universities, research into finger vein recognition is performed and acquisition devices were developed. Examples are the finger vein scanner devices developed by the Civil Aviation University of China [21] and the University of Electronic Science and Technology [9]. The latter device also has the capability of making 3D recordings of finger veins. A more recent sensor, developed at the Norwegian 66 R. Veldhuis et al. Biometrics Laboratory (NBL), allows simultaneous capturing of both finger vein patterns and fingerprints [13]. This is a closed sensor, and the user has to place his finger through a hole inside the device. The device developed at the University of Twente, which is described in the subsequent sections, is also an example of this group of finger vein acquisition devices. The huge advantage of these devices, developed by academics, is that they are usually open devices, the image data is accessible and datasets are made available to the research community. This enables us to evaluate and compare various methods for finger vein recognition. 2.3 University of Twente Finger Vein Capture Device A custom transillumination device type has been designed to capture the finger vascular pattern [18, 19]. This type of capture device has been chosen for its simplicity, robustness and the fact that external light interferences have little influence on the captured images. A downside of this type of capture device is the reduced user convenience because the finger is partially obscured during the capture process. All finger vascular pattern capture devices are based on the fact that blood has a higher absorbency than surrounding tissue in the near-infrared spectrum. A schematic cross section of the capture device can be seen in Fig. 2.2. The USB lightbox is responsible for regulating the individual LED intensities and is encapsulated in the capture device for the ease of portability. The overview also shows the slanted mirror indicated in green and the top plate containing the eight LEDs. The total length of the realised capture device is 50 cm, and the maximum height is 15 cm. The constructed capture device consists of three main components: a light source, a camera and a mirror. These components will be described briefly in the successive paragraphs. Light source This the most important part of the capture device since it determines the intensity of the captured image. Eight SFH4550 near-infrared LEDs produced by Osram with a wavelength of 850 nm are used to transilluminate the finger. This LED type has been chosen because it has a small angle of half intensity, which means more power can be directed into the finger. Each individual LED intensity is regulated using a simple control loop in such a way that a uniform intensity along Fig. 2.2 Schematic cross section of the capture device 2 A High-Quality Finger Vein Dataset Collected … 67 the finger is obtained in the captured image. This control loop is also necessary to cope with varying thicknesses along the finger and between various biometric data subjects. The benefit of this simple control loop can be seen in Fig. 2.3. It clearly shows the over- and underexposure in the non-regulated case. Camera The camera used to capture the images is a BCi5 monochrome CMOS camera with firewire interface produced by C-Cam technologies. The camera has been fitted with a Pentax H1214-M machine vision lens with a focal length of 12 mm. This lens is fitted with a B+W 093 infrared filter which has a cutoff wavelength of 930 nm. The filter is used to block out any interfering visible light. The camera is used in 8-bit mode with a resolution of 1280 × 1024 pixels. Mirror To minimise the height of the capture device, a mirror is used so the camera can be placed horizontally. An NT41-405 first surface mirror produced by Edmund Optics has been used for this purpose. The reason for choosing a first surface mirror is to avoid distortions in the captured image. A conventional mirror has its (a) Eight equal LED intensities (b) LED intensities regulated by control loop Fig. 2.3 Benefit of the control loop to adjust the individual LED intensities Fig. 2.4 Realised finger vascular pattern capture device 68 R. Veldhuis et al. reflective layer protected by glass. The refractive indices of glass and air differ which means distortions will occur in the captured image. The final constructed capture device can be seen in Fig. 2.4. 2.4 Description of Dataset The University of Twente Finger Vein Pattern (UTVP) dataset contains 1440 finger vascular pattern images in total which have been collected from 60 volunteering subjects at our university during the 2011–2012 academic year. Images were captured in two identical sessions with an average time-lapse of 15 days. For each data subject, the vascular pattern of the index, ring and middle finger of both hands has been collected twice at each session. This means that each individual finger has been captured four times in total. The captured images have a resolution of 672 × 380 pixels and have a pixel density of 126 pixels per centimetre (ppcm). The images are stored using the lossless 8-bit greyscale Portable Network Graphics (PNG) format. The percentage of male data subjects was 73%, and the percentage of right-handed data subjects was 87%. The dataset represents a young population with 82% of the data subjects falling in the age range of 19–30, and the remaining data subjects were older than this. A set of sample images from the collected dataset can be seen in Fig. 2.5. The quality of the collected images varies among biometric capture subjects, but the variation in quality of the images from the same biometric capture subject is small. (a) Female, age 24 (b) Male, age 32 (c) Male, age 20 (d) Female, age 31 Fig. 2.5 Sample images of the left-hand ring finger from the collected dataset 2 A High-Quality Finger Vein Dataset Collected … 69 The width of the visible blood vessels ranges from 4–20 pixels which, using a pixel density of 126 pixels per centimetre, corresponds to vessel widths of approximately 0.3–1.6 mm. The pixel density was determined by placing a piece of flat graph paper at exactly the same position as the finger and counting the number of pixels per centimetre in the recorded image. This resulted in a pixel density of 126 pixels per centimetre. The UTVP dataset is available from the University of Twente by completing an online download request and license agreement, see [17]. 2.5 Results 2.5.1 Performance Analysis To illustrate and rank the quality of the collected dataset, the performance of a few published algorithms was evaluated. These algorithms have been applied to our collected dataset and the V4 finger vein database from the Peking University [12] which has been used as a reference. The performance of the algorithms is measured in terms of Equal Error Rate (EER). The experiments also investigate the merit of Adaptive Histogram Equalisation (AHE) as a preprocessing step. Each directory of the Peking dataset contains between four and eight images of the same finger. For the experiments only directories containing exactly eight images have been used, this accounts for 153 directories out of the available 200 directories. For this dataset, it is not known which fingers belong to the same subject. For both datasets, 10% of the fingers have been used for tuning the various parameters of the algorithms. For the Peking dataset, the valid directories are sorted ascending by filename and the first 10% are used for parameter tuning. For our dataset, 10% of the fingers have been selected by taking the first finger of the first data subject, the second finger of the second data subject … the first finger of the seventh data subject. This method of selecting the training set has been chosen to get a larger variation in the quality of the vascular pattern images. The other 90% of both datasets have been used to determine the actual performance of the algorithms. The exact number comparison trials done for both the parameter tuning and the actual determination of the performance are given in Table 2.1. For all of these experiments, fingers were treated as identical individual biometric samples, for example, left-hand index fingers were compared with right-hand middle fingers. Two performance experiments are done per dataset, one with and one without adaptive histogram equalisation as preprocessing step. This preprocessing step is done using MATLAB’s adapthisteq() function with the default parameters’ set. The effect of applying an adaptive histogram equalisation to a vascular pattern image can be seen in Fig. 2.6. To ensure that only image regions containing finger are compared with each other a binary mask is used. This mask is created by first determining the edges of the 70 R. Veldhuis et al. Table 2.1 Number of mated and non-mated comparison trials performed Dataset # fingers Mated Non-mated Parameter tuning Peking 15 UTFVP 35 Actual performance experiment Peking 138 UTFVP 325 420 210 6720 9520 3864 1950 604,992 842,400 (a) Original image (b) Adaptive Histogram Equalisation Fig. 2.6 Effect of Adaptive Histogram Equalisation finger in the image using the method described by Lee et al. [8] and then filling in the area between these edges. The edges detected in the previous step are used to normalise the image using the method described by Huang et al. [5]. This method tries to estimate a rotation and a translation based on the detected finger edges. After these parameters have been estimated, they are used to define an affine image transformation which aligns the finger to the centre of the image. This affine transformation is also applied to the binary mask. The output of each of the algorithms, except the normalised cross-correlation, is a binary template indicating the position of a blood vessel. Two binary templates are compared with each other by using the method described by Miura et al. [10]. An incidental side effect of using the binary finger region mask is that the shape of the finger is also indirectly taken into account when comparing two templates. The final verification results are shown in Table 2.2 which indicates that our dataset performs significantly better in all cases and that adaptive histogram equalisation is beneficial in most of the cases. The results presented here have been independently replicated by Vanoni et al. [20]. The two methods proposed by Miura et al. have been tested by other researchers using their own collected datasets. One of them is Huang et al. [5] who has achieved an EER of 2.8% for the maximum curvature method and an EER of 5% for the repeated line tracking method. Another one is Choi et al. [2] who have achieved an EER of 3.6% for the maximum curvature method. The last one is Kumar and Zhou [7] who achieved an EER of 8.3% for the repeated line tracking method and 2 A High-Quality Finger Vein Dataset Collected … 71 Table 2.2 Performance expressed in terms of EER (%) of several algorithms for both datasets, both with and without Adaptive Histogram Equalisation (AHE) as a preprocessing step EER (%) Original paper Best reported Peking UTFVP No AHE With AHE No AHE With AHE Normalised crosscorrelationa Maximum curvatureb Repeated line trackingc Principal curvatured Wide line detectore 0.0 – 14.7 9.8 3.1 1.9 0.0 2.7e 1.2 1.3 0.4 0.4 0.2 5.0f 6.8 5.9 0.9 1.2 0.4 – 2.7 2.2 0.8 0.4 0.9 – 4.7 2.7 1.5 0.9 a See [6] See [11] c See [10] d See [2] e See [5] f See [7] b achieved an EER of 2.7% for the maximum curvature method. The mentioned EERs from Kumar and Zhou are the average EER of the middle and index fingers. The best reported performance figures for these two methods are mentioned in Table 2.2 as well. Our MATLAB implementation of these algorithms can be found in [16]. 2.6 Next-Generation Finger Vein Scanner 2.6.1 Overview Since the design of the described finger vein scanner, we developed a second version of the finger vein scanner with new capabilities, see Fig. 2.7 [14]. The scanner is much more compact and is built using cheaper components: it uses Raspberry Pi processing boards and cameras. This new scanner has been designed in such a way as to support further research in various ways. It supports multiple NIR LED strips that can be positioned in a semicircle from 0◦ to 180◦ . It also supports three cameras, thus allowing for 3D finger vein reconstruction. Currently, we are investigating optimal illumination and settings of the cameras and 3D finger vein reconstruction. 72 R. Veldhuis et al. Fig. 2.7 Second-generation finger vein scanner of the University of Twente. It has three cameras for 3D recordings and multiple adjustable LED strips 2.6.2 Illumination Control The setup with multiple LED strips that can be rotated up to 180◦ allows for various illumination methods. It supports both transmission and side illumination. Reflection is not supported, however. Care was taken to position the LED strip with respect to the finger position and the opening for the finger with below it the infrared filter, such that as little as possible infrared light can “leak” around the finger. The new setup inherited the advanced control over the intensity of each individual LED from the previous version of the scanner, enabling a more homogeneous illumination and adjustment to the properties of the finger (e.g. thick and thin fingers). In Fig. 2.8, a comparison is made between images recorded using the first- and second-generation finger vein scanner of the University of Twente. The images of the new scanner show much less overexposure at the boundaries of the fingers. We are currently investigating various ways to optimise illumination ranging from illumination from different angles and multiple LED strips to refined control of the LED intensities and combination of multiple images with different illumination. 2.6.3 3D Reconstruction The advantage of 3D recordings is that if fingers are slightly rotated, causing a deformation of the finger vein pattern, this deformation can be compensated for. Another possibility is direct comparison of 3D finger vein patterns. Using the three cameras in the new scanner, we used stereo reconstruction to recover the 3D vein patterns. A preliminary result of 3D finger vein reconstruction using this secondgeneration finger vein scanner is shown in Fig. 2.9 [1]. 2 A High-Quality Finger Vein Dataset Collected … 73 Fig. 2.8 Comparison between images of the same fingers captured by the first (top)- and second (bottom)-generation finger vein scanners. The images captured by the new scanner show less overexposure near the boundaries of the fingers Fig. 2.9 Preliminary 3D finger vein reconstruction using new vein scanner Careful observation shows that the veins at the sides of the fingers are somewhat above the veins in the middle of the finger, i.e. they follow the curvature of the surface of the finger. This is to be expected, because only the veins at the surface of the finger can be visualised using this technique. 2.7 Conclusions A finger vascular pattern dataset containing 1440 high-quality images is presented to the research community. Despite the low number of 60 data subjects which participated, the major contribution of this dataset is the addition of demographic data such as gender, age, and handedness. Another contribution is the high quality of the captured images and the known pixel density of the images. Furthermore, the data 74 R. Veldhuis et al. is collected in two identical sessions with a time lapse of approximately 2 weeks. Because of the high quality of the captured images, our dataset can pave the way for the research of high-security cooperative applications. The performance evaluation using existing algorithms has shown that equal error rates down to 0.4% can be achieved by using our dataset. 2.8 Future Work The use of the vascular pattern of the finger as a biometric is still not as mature as other biometric traits such as 2D face recognition. To reach an equal maturity, more research is needed. Future research should include the collection of larger datasets including 3D data, together with demographic data of the data subjects. These larger datasets will enable researchers to report performance figures with a higher confidence. It will also enable the research of factors such as age, gender and ethnicity on the performance. The research community would also greatly benefit from standardised testing methods and datasets. The biometric performance can further be improved by fusing other finger traits such as traditional fingerprints, the crease pattern of the finger and the shape of the finger. An advantage of finger shape is that it is already present in the captured image. The current control loop which adjusts the LED intensities is still rather crude and leaves space for further improvements in terms of speed and image intensity uniformity. Preliminary results have shown that the relation between the intensity in the captured image and the intensity of the LEDs is as good as linear. Finally, 3D scanning techniques allow compensation of distortions of the finger vein pattern caused by rotation of the finger. Also, direct 3D finger vein comparison is an interesting subject for further research. References 1. Bunda S (2018) 3D point cloud reconstruction based on the finger vascular pattern. Bachelor’s thesis, University of Twente, Netherlands 2. Choi J, Song W, Kim T, Lee S, Kim H (2009) Finger vein extraction using gradient normalization and principal curvature. In: Niel KS, Fofi D (eds) Image processing: machine vision applications II, SPIE-IS&T Electronic Imaging, vol 7251. SPIE. https://doi.org/10.1117/12.810458 3. Hitachi Ltd. (2018) Finger vein reader. http://www.hitachi.co.jp/products/it/veinid/global/ products/embeddeddevicesr.html 4. Hitachi Ltd. (2018) USB finger vein biometric authentication unit. http://www.hitachi.co.jp/ products/it/veinid/global/products/embeddeddevicesu.html 5. Huang B, Dai Y, Li R, Tang D, Li W (2010) Finger-vein authentication based on wide line detector and pattern normalization. In: 20th International conference on pattern recognition, pp 1269–1272. https://doi.org/10.1109/ICPR.2010.316 6. Kono M, Ueki H, Umemura S (2002) Near-infrared finger vein patterns for personal identification. Appl Opt 41(35):7429–7436. https://doi.org/10.1364/AO.41.007429 2 A High-Quality Finger Vein Dataset Collected … 75 7. Kumar A, Zhou Y (2012) Human identification using finger images. IEEE Trans Image Process 21(4):2228–2244. https://doi.org/10.1109/TIP.2011.2171697 8. Lee E, Lee H, Park K (2009) Finger vein recognition using minutia-based alignment and local binary pattern-based feature extraction. Int J Imaging Syst Technol 19(3):179–186. https://doi. org/10.1002/ima.20193 9. 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In: IEEE International joint conference on biometrics, pp 1–7. https://doi.org/10.1109/BTAS.2014.6996225 14. Rozendal S (2017) Redesign of a finger vein scanner. Bachelor’s thesis, University of Twente, Netherlands 15. Sony Corporation (2009) Sony develops compact sized, high speed, high accuracy finger vein authentication technology dubbed “mofiria”. https://www.sony.net/SonyInfo/News/Press/ 200902/09-016E/index.html 16. Ton B (2012, 2017) MATLAB implementation of Miura et al. vein extraction methods. University of Twente, Netherlands. https://www.mathworks.com/matlabcentral/fileexchange/35716miura-et-al-vein-extraction-methods 17. Ton B, Veldhuis R (2012) University of Twente Finger Vascular Pattern (UTFVP) dataset. https://www.utwente.nl/en/eemcs/ds/downloads/utfvp/ 18. Ton BT (2012) Vascular pattern of the finger: biometric of the future? sensor design, data collection and performance verification. Master’s thesis, University of Twente 19. 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https://openalex.org/W2119327257	https://dash.harvard.edu/bitstream/1/26878665/1/2625-06.pdf	English	null	Direct Evidence of Lower Viral Replication Rates In Vivo in Human Immunodeficiency Virus Type 2 (HIV-2) Infection than in HIV-1 Infection	Journal of virology	2,007	cc-by	6,525	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26878665 Citation MacNeil, A., A. D. Sarr, J.-L. Sankale, S. T. Meloni, S. Mboup, and P. Kanki. 2007. “Direct Evidence of Lower Viral Replication Rates In Vivo in Human Immunodeficiency Virus Type 2 (HIV-2) Infection Than in HIV-1 Infection.” Journal of Virology 81 (10) (February 28): 5325–5330. doi:10.1128/jvi.02625-06. Published Version doi:10.1128/JVI.02625-06 Direct Evidence of Lower Viral Replication Rates In Vivo in Human Immunodeﬁciency Virus Type 2 (HIV-2) Infection than in HIV-1 Infection Adam MacNeil,1 Abdoulaye Dieng Sarr,1 Jean-Louis Sankale´,1 Seema Thakore M Souleymane Mboup,2 and Phyllis Kanki1* Adam MacNeil,1 Abdoulaye Dieng Sarr,1 Jean-Louis Sankale´,1 Seema Thakore Meloni,1 Souleymane Mboup,2 and Phyllis Kanki1* Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts,1 and Laboratoire de Bacteriologie et Virologie, Universite Cheikh Anta Diop, Dakar, Senegal2 Souleymane Mboup,2 and Phyllis Kanki1* Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts,1 and Laboratoire de Bacteriologie et Virologie, Universite Cheikh Anta Diop, Dakar, Senegal2 Received 28 November 2006/Accepted 20 February 2007 Studies have shown that human immunodeﬁciency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantiﬁed total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-naı¨ve HIV-1-infected (n 21) and HIV-2-infected (n 18) individuals from Dakar, Senegal, with CD4 T-cell counts of >200/l. Consistent with our previous ﬁndings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumu- lation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo. linear unintegrated form (8), while integrated proviral DNA accounts for a minor fraction of the total DNA load in vivo (6, 8, 9, 15, 16). Two closely related human lentiviruses, human immunode- ﬁciency virus type 1 (HIV-1) and HIV-2, have been shown to cause AIDS. * Corresponding author. Mailing address: Department of Immunol- ogy and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave., Boston, MA 02115. Phone: (617) 432-1267. Fax: (617) 432-3575. E-mail: pkanki@hsph.harvard.edu. Published ahead of print on 28 February 2007. Direct Evidence of Lower Viral Replication Rates In Vivo in Human Immunodeﬁciency Virus Type 2 (HIV-2) Infection than in HIV-1 Infection However, it is now well recognized that the in vivo pathogenicity of HIV-2 is attenuated relative to that of HIV-1, with signiﬁcantly lower rates of disease progression and trans- mission (20, 27, 28, 43). Consistent with these observations, plasma viral loads are signiﬁcantly lower in people infected with HIV-2 (1, 2, 36, 37, 42). Interestingly, studies have shown that quantities of total viral DNA in peripheral blood mono- nuclear cells (PBMCs) are similar in people infected with HIV-1 and people infected with HIV-2 (5, 32, 36). ( ) While higher plasma viral loads in HIV-1 infection suggest higher replication rates, no studies have clearly shown a dif- ference in replication in vivo between HIV-1 and HIV-2. Ad- ditionally, previous studies comparing viral DNA in HIV-1 and HIV-2 infection have focused on total viral DNA and, to date, no studies have quantiﬁed integrated proviral DNA in people infected with HIV-2. In this study, we measured viral life cycle intermediates (integrated proviral DNA and viral mRNA) in conjunction with total viral DNA loads and plasma viral loads in people infected with HIV-1 or HIV-2 to determine whether quantitative differences that can explain the lower plasma viral loads observed in HIV-2 infection exist at a speciﬁc point of the viral life cycle. Within an infected cell, HIV DNA is found in a number of forms, including integrated proviral DNA, unintegrated linear viral DNA, and unintegrated one- and two-long-terminal-re- peat (LTR) episomal viral DNA (31). The integration of pro- viral DNA is necessary for viral replication (10, 11, 21, 44). In addition, the integration of proviral DNA can result in a stable long-term infection. HIV-1 has been shown to establish latent infection in vivo within resting memory CD4 T cells, a reser- voir in which the integrated proviral genome can persist for decades (reviewed in references 22 and 35). Although integra- tion is necessary for viral replication, studies of HIV-1 have shown that the majority of in vivo viral DNA exists in the Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility JOURNAL OF VIROLOGY, May 2007, p. 5325–5330 0022-538X/07/$08.000 doi:10.1128/JVI.02625-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Vol. 81, No. 10 JOURNAL OF VIROLOGY, May 2007, p. 5325–5330 0022-538X/07/$08.000 doi:10.1128/JVI.02625-06 Copyright © 2007, American Society for Microbiol RESULTS For this study, we examined 21 subjects infected with HIV-1 and 18 subjects infected with HIV-2 from Dakar, Senegal. All had CD4 T-cell counts above 200/l and were antiretroviral therapy naı¨ve. Although all subjects were in the asymptomatic phase of infection, CD4 T-cell counts were lower (P 0.0384) in HIV-1-infected subjects (median and interquartile range values were 560 and 404 to 614 cells/l, respectively [for HIV-2-infected subjects, median and interquartile range val- ues were 946 and 534 to 1,503 cells/l, respectively]). In addi- tion, HIV-1-infected subjects (median and interquartile range values were 34.9 and 27.4 to 42.2 years, respectively) were younger (P 0.0039) than HIV-2-infected subjects (median and interquartile range values were 44.2 and 40.1 to 48.9 years, respectively). Quantiﬁcation of integrated proviral DNA. Quantities of integrated proviral DNA were determined for HIV-1 and for HIV-2 by using an endpoint dilution Alu-PCR approach as previously described (9). A series of six threefold serial dilutions were performed in duplicate from a starting quantity of 1 g genomic DNA, and Alu-PCR was performed using primers Alu1 (GCCTCCCAAAGTG CTGGGATTACAG) (34) and AM1C1r (CTTAATACTGACGCTCTCGCA CCC) for HIV-1 samples and Alu1 and AM2C1r (AAGGGTCCTAACAGAC CAGGGTCT) for HIV-2 samples in a total volume of 100 l under the following reaction conditions: denaturation at 94° for 5 min; 35 PCR cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 3 min; and a ﬁnal extension at 72°C for 7 min. The ampliﬁcation of integrated proviral DNA was assayed by real-time PCR with 5 l of the ﬁrst-round Alu-PCR product by using previously described HIV-1 and HIV-2 real-time PCR assays (25). Real-time PCR assays were performed for each sample in parallel with equivalent concentrations of nonampliﬁed genomic DNA from that respective sample to control for nonampliﬁed viral DNA carryover from the Alu-PCR. Integrated proviral DNA copy numbers were determined by using the QUALITY program (38). Previous studies have indicated that plasma viral loads are higher in people infected with HIV-1 than in people infected with HIV-2 (1, 2, 36, 37, 42), despite their having similar amounts of total viral DNA (5, 32, 36). For this study, we quantiﬁed total viral DNA by using real-time PCR assays for HIV-1 and HIV-2 that ampliﬁed highly conserved regions of the viral genomes that correspond to the LTR-gag junction. Twenty of 21 HIV-1-infected and 17 of 18 HIV-2-infected individuals had detectable total viral DNA. MATERIALS AND METHODS Sample acquisition. PBMC samples were obtained from a cohort of female sex workers in Dakar, Senegal, that have been followed since 1985. Epidemiologic and clinical aspects of this cohort have previously been described elsewhere (19). All subjects signed informed consents and participated in protocols approved by the Counseil National de Lutte Contre le Sida Comite Ethique et Juridique and the Harvard School of Public Health Human Subjects Committee. CD4 T-cell counts were determined, and serum samples were diagnosed for HIV-1- and HIV-2-speciﬁc antibodies as previously described (19). All subjects enrolled in this study were antiretroviral therapy naı¨ve and had CD4 T-cell counts above 200/l at the time of sample acquisition. DNA and mRNA extraction. Cryopreserved PBMC samples were thawed and rested overnight in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum and 1% antibiotics to ensure sample viability and remove dead 5325 J. VIROL. MACNEIL ET AL. 5326 tiﬁed relative to the full-length HIV-2 genomic RNA standard curve in a two- step, real-time reverse transcription-PCR, as described above. tiﬁed relative to the full-length HIV-2 genomic RNA standard curve in a two- step, real-time reverse transcription-PCR, as described above. cells. The following day, PBMC samples were divided equally, with half used for the extraction of DNA and half used for the extraction of mRNA. DNA was extracted (blood and cell culture DNA mini kit; QIAGEN), and DNA concen- trations were determined by an optical density reading at 260 nm. Polyadenylated mRNA was extracted (Oligotex mRNA direct mini kit; QIAGEN) and immedi- ately converted to cDNA (TaqMan reverse transcription reagents; Applied Bio- systems) by using an equal-volume mixture of random hexamer and poly(T) primers. Statistical analyses. Statistical analyses were performed using SAS 9.1. For analytical purposes, HIV-2-infected subjects with plasma viral loads of 400/ml were assigned values of 400/ml in accordance with the detection limit of the HIV-1 plasma viral load assay (Amplicor HIV-1 monitor test, version 1.5; Roche). The Wilcoxon rank sum test was used for an unadjusted comparison of CD4 T-cell counts, age, total viral DNA, integrated proviral DNA, viral mRNA, plasma viral load, and percentage of total viral DNA integrated between HIV-1- and HIV-2-infected subjects; two-sided P values are re- ported. Because of the limited number of study subjects available, we were unable to directly match HIV-1-infected and HIV-2-infected individuals on the basis of CD4 T-cell count and age. RESULTS Among study sub- jects, total viral DNA loads were slightly higher in HIV-1- infected subjects than in HIV-2-infected subjects (Fig. 1). However, after adjusting for CD4 T-cell counts and age, there was no statistical difference in total viral DNA levels between HIV-1- and HIV-2-infected subjects. In contrast, in both unadjusted and adjusted analyses, plasma viral loads were signiﬁcantly higher in HIV-1-infected subjects than those in HIV-2-infected subjects. Quantiﬁcation of plasma viral loads. HIV-1 plasma viral loads were deter- mined by using a commercially available assay (Amplicor HIV-1 monitor test, version 1.5; Roche). To determine HIV-2 viral loads, we developed an in-house real-time PCR viral load assay. Brieﬂy, we ampliﬁed a fragment of HIV-2 LTR-gag from a previously cloned HIV-2 DNA sample using primers HIV2gagF and AM2gag1r and synthesized a T7 promoter-driven in vitro transcription construct by using commercially available reagents (BLOCK-iT RNAi TOPO transcription kit; Invitrogen) and primer AM2gag1r. HIV-2 RNA was generated by in vitro transcription (MEGAshortscript; Ambion). In vitro-transcribed HIV-2 RNA was puriﬁed (MEGAclear; Ambion), treated with DNase (New England Biolabs), and repuriﬁed, and the concentration of in vitro-transcribed HIV-2 RNA was determined by an optical density reading at 260 nm. To create a full-length HIV-2 genomic RNA standard, full-length HIV-2 genomic RNA from a previously described in vitro infection (25) was extracted (QIAamp viral RNA mini kit; QIAGEN), treated with DNase (New England Biolabs), and repuriﬁed (MEGAclear; Ambion). Quantities of full-length HIV-2 genomic RNA were determined by using the in vitro-transcribed HIV-2 RNA as a quan- titative standard in a two-step, real-time reverse transcription-PCR (TaqMan reverse transcription reagents and TaqMan universal PCR master mix; Applied Biosystems) using the primer HIV2gagR for the cDNA synthesis reaction and the HIV-2 LTR-gag real-time PCR primers and probe described above. For the quantiﬁcation of plasma viral loads, RNA standard curves were generated from the full-length HIV-2 genomic RNA standard, spiked to noninfected lysis buffer, and extracted in parallel with 280 l of plasma from HIV-2-infected subjects (QIAamp viral RNA mini kit; QIAGEN). HIV-2 plasma viral loads were quan- In order to compare amounts of viral mRNA between HIV-1 and HIV-2, viral transcripts isolated though the extrac- tion of polyadenylated cellular mRNA were quantiﬁed using the same real-time PCR assays that were used to quantify total viral DNA. MATERIALS AND METHODS Alternatively, to adjust for CD4 T-cell counts and age in the comparison of HIV-1-infected and HIV-2- infected individuals, regressions were performed using the ROBUSTREG procedure, which provides stable results in the presence of outlying values, and values of total viral DNA, integrated proviral DNA, viral mRNA, and plasma viral loads were log transformed. Correlations of total viral DNA, integrated proviral DNA, percentage of total DNA integrated, and viral mRNA with plasma viral loads were examined using the Pearson’s correlation coefﬁcient (for HIV-1) and Spearman’s correlation coefﬁcient (for HIV-2) by using log-transformed values of total viral DNA, integrated proviral DNA, viral mRNA, and plasma viral loads. Quantiﬁcation of total viral DNA and viral mRNA. HIV-1 total viral DNA and mRNA were quantiﬁed using a previously described real-time PCR assay (25) that ampliﬁes a highly conserved region corresponding to the HIV-1 LTR-gag junction. For the quantiﬁcation of HIV-2 total viral DNA and mRNA, we developed a novel HIV-2 real-time PCR assay that ampliﬁes a highly conserved region corresponding to the HIV-2 LTR-gag junction. To construct a quantiﬁ- cation standard, a fragment of HIV-2 LTR-gag was ampliﬁed by PCR from a previously cloned HIV-2 DNA sample using primers AM2gag1f (AGACCCTG GTCTGTTAGGACCCTT) and AM2gag1r (CAATTCATTCGCTGCCCA CAC) cloned into an expression vector (pCR2.1; Invitrogen) and transformed into competent cells (TOP10; Invitrogen). Cloned plasmid was puriﬁed (SNAP MiniPrep kit; Invitrogen), and the concentration of puriﬁed plasmid was deter- mined by an optical density reading at 260 nm. For the quantiﬁcation of HIV-2 samples, real-time PCRs were performed by using primers HIV2gagF (CCAA CCACGACGGAGTGCTC) and HIV2gagR (CTCTCAAGACGGAGTTTCT CGC) and detected by using the probe HIV2gagP (AGGCCTCCGGGTGAAG GTAAG), which contained a 5 6-carboxyﬂuorescein ﬂuorescent reporter and a 3 MGB nonﬂuorescent quencher. For HIV-1 and HIV-2 real-time PCR assays, real-time PCR reagents and reaction conditions were as previously described (25). Viral mRNA was standardized to cellular beta-actin mRNA. Beta-actin mRNA was quantiﬁed using commercially available reagents (TaqMan B-actin detection reagents; Applied Biosystems) according to manufacturer recommen- dations. RESULTS Unlike for HIV-1-infected subjects, in which viral transcripts were detectable for the majority of subjects, amounts of viral mRNA were below the limit of detection for LOW HIV-2 REPLICATION RATE IN VIVO 5327 VOL. 81, 2007 FIG. 1. Total viral DNA, integrated proviral DNA, viral mRNA, and plasma viral loads for HIV-1-infected and HIV-2-infected subjects. Units refer to copies per 106 PBMCs (total viral DNA and integrated proviral DNA), copies per 105 copies beta-actin (viral mRNA), and viral load per milliliter of plasma (plasma viral load). Unadjusted P values refer to Wilcoxon rank sum two-sided P values. Adjusted P values refer to robust regression P values adjusted for CD4 T-cell count and age. Differences between HIV-1 and HIV-2 that reach statistical signiﬁcance are in bold. FIG. 1. Total viral DNA, integrated proviral DNA, viral mRNA, and plasma viral loads for HIV-1-infected and HIV-2-infected subjects. Units refer to copies per 106 PBMCs (total viral DNA and integrated proviral DNA), copies per 105 copies beta-actin (viral mRNA), and viral load per milliliter of plasma (plasma viral load). Unadjusted P values refer to Wilcoxon rank sum two-sided P values. Adjusted P values refer to robust regression P values adjusted for CD4 T-cell count and age. Differences between HIV-1 and HIV-2 that reach statistical signiﬁcance are in bold. FIG. 1. Total viral DNA, integrated proviral DNA, viral mRNA, and plasma viral loads for HIV-1-infected and HIV-2-infected subjects. Units refer to copies per 106 PBMCs (total viral DNA and integrated proviral DNA), copies per 105 copies beta-actin (viral mRNA), and viral load per milliliter of plasma (plasma viral load). Unadjusted P values refer to Wilcoxon rank sum two-sided P values. Adjusted P values refer to robust regression P values adjusted for CD4 T-cell count and age. Differences between HIV-1 and HIV-2 that reach statistical signiﬁcance are in bold. most HIV-2-infected individuals. The difference in quantities of viral mRNA between HIV-1 and HIV-2 were statistically signiﬁcant in both unadjusted and adjusted analyses. quantities of total viral DNA, integrated proviral DNA, and viral mRNA all signiﬁcantly correlated with plasma viral loads. In contrast, for HIV-1, there was no correlation between the percentage of total viral DNA integrated and plasma viral loads. Among HIV-2-infected subjects, we observed no signif- icant correlation between plasma viral loads and total viral DNA or integrated proviral DNA. RESULTS However, despite the small number of HIV-2-infected subjects having detectable mRNA, quantities of viral mRNA positively correlated with plasma viral loads among HIV-2-infected subjects. As with HIV-1, there was no relationship between the percentage of total viral DNA integrated and plasma viral loads. In order to compare the amounts of integrated proviral DNA in PBMCs of subjects infected with HIV-1 with those of subjects infected with HIV-2, we used a previously de- scribed endpoint dilution Alu-PCR approach (9) that selec- tively ampliﬁes integrated proviral DNA by using a virus- speciﬁc primer and a primer speciﬁc to conserved sequences in Alu elements. Alu elements are retrotransposons, and approximately 1.2 million copies of Alu elements are found in the human genome, accounting for 10 to 11% of the entire genome (18). To avoid unequal ampliﬁcation between HIV-1 samples and HIV-2 samples, HIV-1 and HIV-2 prim- ers were approximately equidistant from the 5 end of the respective viral genomes, and have previously been shown to amplify integrated proviral DNA (25). Twenty of 21 HIV- 1-infected subjects and 16 of 18 HIV-2-infected subjects had detectable integrated proviral DNA. Amounts of integrated proviral DNA were larger for HIV-1 than HIV-2, and values were slightly different after adjusting for CD4 T-cell counts and age. FIG. 2. Percentage of total DNA in the integrated form for HIV- 1-infected and HIV-2-infected subjects. Values are plotted for all sub- jects who had detectable integrated proviral DNA (n 20 for HIV-1; n 16 for HIV-2). Unadjusted P values refer to Wilcoxon rank sum two-sided P values. Adjusted P values refer to robust regression P values adjusted for CD4 T-cell count and age. Previous studies of HIV-1 indicate that only a small propor- tion of total viral DNA is integrated in vivo (6, 8, 9, 15, 16). Among study subjects with detectable quantities of integrated proviral DNA, we observed similar results (Fig. 2) for both HIV-1 (the median percentage of total DNA integrated was 9.2%) and HIV-2 (the median percentage of total DNA inte- grated was 3.1%). There was no statistically signiﬁcant differ- ence in the percentage of DNA integrated between HIV-1- infected subjects and HIV-2-infected subjects. FIG. 2. Percentage of total DNA in the integrated form for HIV- 1-infected and HIV-2-infected subjects. Values are plotted for all sub- jects who had detectable integrated proviral DNA (n 20 for HIV-1; n 16 for HIV-2). RESULTS Unadjusted P values refer to Wilcoxon rank sum two-sided P values. Adjusted P values refer to robust regression P values adjusted for CD4 T-cell count and age. We examined the relationship of total viral DNA, integrated proviral DNA, percentage of total DNA integrated, and viral mRNA to plasma viral loads for HIV-1-infected subjects and HIV-2-infected subjects (Table 1). As expected, for HIV-1, J. VIROL. 5328 5328 MACNEIL ET AL. MACNEIL ET AL. TABLE 1. Correlation of total viral DNA, integrated proviral DNA, percentage of total DNA integrated, and viral mRNA, with plasma viral loads for HIV-1-infected subjects and HIV-2-infected subjects Virus Correlation statistics for viral load anda: Total viral DNA Integrated proviral DNA % of total DNA integrated Viral mRNA r P r P r P r P HIV-1 0.56 0.0088 0.53 0.0130 0.16 0.4900 0.75 0.0001 HIV-2 0.39 0.1091 0.22 0.3837 0.02 0.9371 0.49 0.0389 TABLE 1. Correlation of total viral DNA, integrated proviral DNA, percentage of total DNA integrated, and viral mRNA, with plasma viral loads for HIV-1-infected subjects and HIV-2-infected subjects (14) reported extremely little accumulation of processive viral mRNA in latently infected resting CD4 T cells during HIV-1 infection in vivo. Given the observations that HIV-2 is able to establish integrated proviral infection, yet transcription re- mains limited, HIV-2 may have a much higher propensity for establishing latent infection in vivo than HIV-1 does. In sup- port of this notion, the Nef protein of HIV-2 has been shown to downmodulate T-cell receptor-CD3 within infected cells and block responsiveness to T-cell activation, whereas the HIV-1 Nef protein fails to do so, contributing to increased cell death in HIV-1-infected cells and possibly increased persis- tence in HIV-2-infected cells (40). a Correlations were examined using log-transformed total viral DNA, inte- grated proviral DNA, viral mRNA, and plasma viral loads. Correlation coef- ﬁcients (r) and P values refer to Pearson’s correlation coefﬁcient (for HIV-1) and Spearman’s correlation coefﬁcient (for HIV-2). Correlations that reach statistical signiﬁcance are in bold. Among HIV-1-infected individuals, total viral DNA, inte- grated proviral DNA, and viral mRNA all correlated with plasma viral loads. Of these measures, viral mRNA had the strongest correlation with plasma viral load. Interestingly, de- spite the small number of individuals with detectable mRNA, viral mRNA also correlated with plasma viral load for HIV-2. These ﬁndings support the notion that viral mRNA is a rele- vant marker of viral replication. DISCUSSION In HIV-1 infection, higher plasma viral loads are predictors of more rapid disease progression (3, 23, 29, 30, 33). Plasma viral loads have been clearly demonstrated to be lower in HIV-2 infection than in HIV-1 infection (1, 2, 36, 37, 42), possibly explaining the lower rate of disease progression of HIV-2 (12). This observation, in conjunction with similar total viral DNA loads in people infected with these viruses (5, 32, 36), suggests that HIV-2 maintains a lower rate of replication in vivo. However, host factors, such as differences in adaptive immune responses, could also account for the difference in plasma viral loads between these viruses. To address the hy- pothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantiﬁed replication intermediates in the viral life cycles of HIV-1 and HIV-2 in vivo. Our data suggest that HIV-2 is able to establish a stable integrated proviral infection within PBMCs and that replica- tion is attenuated at some point following integration, but prior to the accumulation of late transcripts. The reason for the block remains unknown. HIV-1 and HIV-2 have different reg- ulatory elements within their promoter regions and have been shown to respond differently to cellular stimuli (13), suggesting possible differences in transcriptional initiation between these viruses. In addition, integration within heterochromatin has been shown to be associated with transcriptional inhibition and viral latency during in vitro HIV-1 infection (17, 24) and we have recently reported evidence of proviral integration within heterochromatin in a higher proportion of people infected with HIV-2 than with HIV-1 (25). Differences between HIV-1 and HIV-2 may also exist following transcriptional initiation. For instance, the HIV-2 LTR is signiﬁcantly larger than the HIV-1 LTR, has been shown to undergo 5 LTR splicing, and has a complex secondary structure that may affect transcriptional trans-activation (4, 7). While the exact effect this has on mRNA accumulation in vivo remains unknown, it is possible that dif- ferences in the LTR structure result in less-efﬁcient transcrip- tional elongation and/or the switch from early to late tran- scripts in HIV-2 infection. Consistent with previous reports, we observed signiﬁcantly higher plasma viral loads in people infected with HIV-1 than in people infected with HIV-2, and after an adjustment for CD4 T-cell counts and age, we observed no difference in total viral DNA. RESULTS For both HIV-1 and HIV-2, no correlation was observed between the percentage of total viral DNA integrated and plasma viral load, indicating that the proportion of integrated proviral DNA is not a pertinent mea- sure for viral pathogenesis. DISCUSSION The interpretation of total viral DNA with regard to viral replication is difﬁcult, as studies have shown that only a minority of HIV-1 DNA is actually integrated in vivo (6, 8, 9, 15, 16). Furthermore, this measurement includes one- and two-LTR circles, which are dead-end products. To better com- pare the nature of viral DNA between HIV-1 and HIV-2 in vivo, we quantiﬁed levels of integrated proviral DNA. We observed only modestly higher levels of integrated proviral DNA in HIV-1-infected individuals, and after comparing the percentage of viral DNA in the integrated form, we observed no difference between HIV-1 and HIV-2. While integrated proviral DNA clearly represents a minor proportion of the overall viral DNA load in HIV-2 infection, our data indicate that the integration of viral DNA is not overtly impaired in HIV-2 infection relative to that in HIV-1. To our knowledge, this is the ﬁrst study to report direct evidence of a difference in the replication rates of HIV-1 and HIV-2 in vivo. In a recent report from our lab, we prospectively examined viral evolution over a decade of infection among eight HIV-2-infected individuals and observed signiﬁcantly lower rates of viral evolution for HIV-2 than for HIV-1 (26), which also supports the notion that HIV-2 replication is atten- uated in vivo in comparison to HIV-1 replication. It should be noted that the results of the current study do not exclude the possibility that differences in adaptive immune responses may also contribute to the lower plasma viral loads observed with HIV-2 infection. Recent data from our laboratory indicate a The HIV-1 and HIV-2 real-time PCR assays used in this study to quantify viral mRNA amplify a fragment of the LTR- gag junction, which is encoded in processive unspliced viral mRNA. Levels of viral mRNA in HIV-2-infected individuals were signiﬁcantly lower than those in HIV-1-infected individ- uals. This difference is consistent with the lower plasma viral loads among the HIV-2-infected individuals and implies that lower plasma viral loads can be accounted for by lower viral mRNA levels in HIV-2 infection in vivo. Hermankova et al. VOL. 81, 2007 5329 LOW HIV-2 REPLICATION RATE IN VIVO broad neutralizing response in people infected with HIV-2 (39). macrophages by human immunodeﬁciency virus type 1. J. Virol. 69:3216– 3219. 12. 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W4284895664.txt	https://downloads.hindawi.com/journals/geofluids/2022/9947113.pdf	en		Deformation and Failure Law of Roadway along Goaf and Reserve Width of Section Coal Pillar	Geofluids	2,022	cc-by	7,787	Hindawi Geoﬂuids Volume 2022, Article ID 9947113, 15 pages https://doi.org/10.1155/2022/9947113 Research Article Deformation and Failure Law of Roadway along Goaf and Reserve Width of Section Coal Pillar Yunsheng Chen, Jian Sun , Di Pang, and Ruofei Zhang State Key Laboratory of Mining Response and Disaster Prevention and Control in Deep Coal Mines, Anhui University of Science and Technology, Huainan 232001, China Correspondence should be addressed to Jian Sun; sj323@cumt.edu.cn Received 3 April 2022; Accepted 18 June 2022; Published 7 July 2022 Academic Editor: Liang Xin Copyright © 2022 Yunsheng Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. It was a non-negligible problem in the reserve of section coal pillar in coal mining. Reasonable section coal pillar width and support parameters can not only ensure the stability of surrounding rock of roadway but also produce more coal resources and save support cost. In this paper, the mining of 9106 workface of Linfen Tianyu Hengsheng Coal Industry of Wanbei Coal Power Group was taken as the research engineering background. FLAC3D numerical simulation software was used to simulate and analyze the laws of deformation failure of 6 m, 7 m, 8 m, 9 m, and 10 m coal pillars and surrounding rock of roadway along goaf without support conditions during the mining of 9106 workface. On this basis, the deformation failure law of 7 m and 8 m coal pillars and surrounding rock of roadway along goaf under diﬀerent supporting conditions (bolt and cable with diﬀerent row spacing) were further simulated and analyzed. The results showed that when 7 m coal pillars were reserved in 9106 roadway along goaf, the optimal bolt row spacing was 800 mm. When 8 m coal pillars were reserved, the bolt row spacing was preferred to be 900 mm. At this time, the width of coal pillar and support parameters can ensure the stability of roadway surrounding rock during the mining process of 9106 workface and can produce more coal resources and save support cost. 1. Introduction The section coal pillar is a problem that cannot be ignored in coal mining, and the width of coal pillar plays a vital role in the safe mining of workface. If a large coal pillar is reserved to protect the roadway, the strength of coal pillar is high and the bearing capacity is strong, which is beneﬁcial to the stability and maintenance of the surrounding rock of the roadway. However, there are more coal resources left, which reduces the coal recovery rate. If a narrow coal pillar is reserved, it is easy to cause coal pillar instability. Due to the inﬂuence of dynamic pressure in fully mechanized caving mining, the distribution range of lateral abutment pressure will increase, and the deformation and failure of roadway surrounding rock will be serious, resulting in diﬃculty in roadway maintenance [1–8]. Reasonable width of section coal pillar and support parameters can not only ensure the stability of roadway surrounding rock but also extract more coal resources and save support costs. Therefore, it is of great scientiﬁc signiﬁcance and engineering application value to study the deformation and failure law of roadway along goaf and the width of section coal pillar [9–15]. Many scholars have made many research results on the deformation and failure law of roadway along goaf and the width of section coal pillars. Zhao et al. [16] used theoretical analysis, numerical simulation, and ﬁeld test methods to study the deformation and failure characteristics of roadway surrounding rock in fully mechanized caving workface, which showed that the stability of coal pillar was mainly aﬀected by the main roof fracture and multiple mining activities. With the increase of coal pillar width, 2 Geoﬂuids 80 11 70 11 9106 workface 60 11 50 11 1060 11 40 11 1040 1050 2C–3 30 11 20 11 10 11 00 9016 0 109 0 108 0 107 9102 goaf 0 106 0 105 0 103 0 104 2C–3 X4018426. 09 Y37527980. 88 30 10 9104 11 50 910 20 40 10 11 7 113 10 0 10 0 110 0 111 00 07 0 112 2 10 91 Figure 1: Coal seam mineral component testing. the stress concentration area is transferred from the middle of the solid coal side to the middle of the coal pillar, and the plastic failure area shows a “X” penetration trend from both sides to the middle of the coal pillar. Zhang et al. [17] took 8407 fully mechanized caving workface of Yangquan No. 5 Coal Mine as an example, based on the basic roof fracture mechanics model of the goaf and the limit equilibrium theory of surrounding rock, calculated the upper and lower limits of reasonable coal pillar width. And using the borehole stress monitoring method, the internal stress distribution of the coal pillar during mining process of the 8407 fully mechanized caving workface was measured, and then the support parameters of the roadway along the gob were determined. Based on in-situ stress measurement and three-dimensional modeling technology, Jiang et al. [18] analyzed the vertical stress distribution characteristics of the coal roof at diﬀerent positions on the edge of the 15111 workface in the tectonic province, as well as the vertical stress on the coal pillars of diﬀerent widths, the horizontal stress of the roadway roof, and the deformation of the surrounding rock of roadway, to explore a method to determine the reasonable coal pillar width along the goaf roadway in the tectonic stress zone. Li et al. [19] made statistics of 6 typical coal mine cases in eastern and western China, analyzed the key factors aﬀecting the deformation and failure of narrow coal pillar, and studied the deformation and failure characteristics of narrow coal pillar under the inﬂuence of diﬀerent coal seam strength and thickness, coal seam buried depth, basic roof strength, tunneling, mining, and support strength by using FLAC numerical simulation, and put forward the control countermeasures to ensure the stability of narrow coal pillar under diﬀerent inﬂuencing factors. Based on the engineering background of 1310 workface under the strip coal pillar in Jinqiao Coal Mine, through theoretical analysis, similar simulation, and numerical simulation, Yang et al. [20] studied the stress distribution law, plastic failure range, and rationality of coal pillar setting in diﬀerent width sections, and determined the reasonable roadway position of workface under strip coal pillar in rock burst mine. In order to improve the recovery rate of coal resources, many mines generally use narrow coal pillars. However, due to the mining impact of workface, the deformation and failure of roadway surrounding rock are serious. Especially when one side of the workface is mined, the deformation and failure of surrounding rock of roadway along goaf are more serious, and roadway maintenance is diﬃcult [21–24]. Therefore, based on the engineering background of 9106 workface of Linfen Tianyu Hengsheng Coal Industry of Wanbei Coal Power Group, the numerical simulation method was used to study and analyze the deformation and failure law of 9106 roadway along goaf (Machine lane), and the width of coal pillars and supporting parameters. The research results can ensure the stability of the surrounding rock of roadway along goaf during the mining of 9106 workface, at the same time, more coal resources can be mined and the support cost can be saved. 2. Engineering Situation The 9106 workface of Hengsheng Coal Industry is located in the north of ﬁrst mining area of mine, and the west of 9106 workface is adjacent to the boundary of mining area and the north of the third mining area 9301 goaf, and the east of the 9102 goaf, south of the east wing track downhill. The 9106 Geoﬂuids 3 9106 workface 9102 goaf Roadway along goaf Section coal pillar Figure 2: Numerical calculation model of 9106 workface. Table 1: Physical and mechanical parameters of roof and ﬂoor of 9106 workface. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Lithology Overburden strata Sandstone K4 ﬁne sandstone Fine sandstone Mudstone K3 limestone Mudstone K2 limestone Coal seam Mudstone Aluminum mudstone K1 ﬁne sandstone Fine sandstone Mudstone Limestone Underlying strata Bulk modulus/ Shear modulus/ Tensile strength/ GPa GPa MPa Cohesion/ MPa Internal friction angle/° 3.30 1.85 30 1.20 1.80 1.17 28 16.9 6.00 1.80 2.46 35 2650 2650 2650 2650 2650 1400 2650 9.0 1.50 16.9 1.50 16.9 2.40 1.50 3.20 0.50 6.00 0.50 6.00 1.07 0.50 1.80 1.30 1.80 1.30 1.80 0.60 1.30 2.87 1.55 2.46 1.55 2.46 1.26 1.55 32 30 35 30 35 27 30 4.0 2650 2.25 1.40 1.60 1.42 30 4.0 2650 9.00 3.20 1.80 2.87 32 4.0 6.0 8.0 50.0 2650 2650 2650 2650 9.00 1.50 9.00 29.3 3.20 0.50 3.20 12.4 1.80 1.30 1.80 3.30 2.87 1.55 2.87 4.20 32 30 32 42 Thickness/ m Density/ kg.m-3 59.0 2650 1.90 0.90 12.0 2650 4.0 2.0 2650 4.0 8.0 4.0 4.0 15.0 5.0 8.0 workface is 712 m length and 202 m width, with a mining area of 143918 m2. The dip angle of mining No. 9 + 10 coal seam is 3 ~ 9°, with an average dip angle of 7°. The occurrence elevation of coal ﬂoor is 1095 ~ 1190 m, the thickness of coal seam is 4.9 ~ 8.1 m, and the average coal thickness is 6.5 m (net coal thickness is 5.1 m). The direct roof of 9106 workface is dominated by K2 limestone, with a stable thickness of 13.1 m on average. The compressive strength is large, and cracks are developed and compact and hard. The direct bottom of 9106 workface is mainly mudstone, with an average thickness of 10.8 m, belonging to ﬂoppy disk rock, and partially bauxite. The engine lane of 9106 workface is roadway along goaf, which makes the 9106 engine lane be aﬀected by the mined-out area of 9102 and the workface in the mining process of 9106 workface, as shown in Figure 1. Therefore, it is necessary to study the deformation and failure law of 9106 roadway along goaf (engine roadway) and the width of section coal pillar to ensure the safety of 9106 workface mining. 3. Numerical Calculation Model Based on the hydrogeological data of Hengsheng Coal Industry and the comprehensive geological histogram of 9106 workface, FLAC3D numerical calculation software was used to establish the mining numerical calculation model of 9106 workface as shown in Figure 2. The length, width, and height of the model are 774 m, 566 m, and 200 m, respectively. The numerical calculation model adopted Mohr-Coulomb yield criterion, including 9102 4 Geoﬂuids FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Contour of ZZ-stress Plane: on Calculated by: volumetric averaging Plane: on Calculated by: volumetric averaging 1.7436E+05 2.0247E+05 0.0000E+00 0.0000E+00 –2.0000E+06 –2.0000E+06 –4.0000E+06 –4.0000E+06 –6.0000E+06 –6.0000E+06 –8.0000E+06 –8.0000E+06 –1.0000E+07 –1.0000E+07 –1.2000E+07 –1.2000E+07 –1.4000E+07 –1.4000E+07 –1.6000E+07 –1.8000E+07 –1.6000E+07 –2.0000E+07 –1.8000E+07 –2.2000E+07 –2.0000E+07 –2.2189E+07 –2.1989E+07 (a) 6 m coal pillar (b) 7 m coal pillar FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Contour of ZZ-stress Plane: on Calculated by: volumetric averaging Plane: on Calculated by: volumetric averaging 2.9897E+05 3.3560E+05 0.0000E+00 0.0000E+00 –2.0000E+06 –2.0000E+06 –4.0000E+06 –4.0000E+06 –6.0000E+06 –6.0000E+06 –8.0000E+06 –8.0000E+06 –1.0000E+07 –1.0000E+07 –1.2000E+07 –1.2000E+07 –1.4000E+07 –1.4000E+07 –1.6000E+07 –1.6000E+07 –1.8000E+07 –1.8000E+07 –2.0000E+07 –2.0000E+07 –2.1495E+07 –2.1086E+07 (c) 8 m coal pillar (d) 9 m coal pillar FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Plane: on Calculated by: volumetric averaging 3.5730E+05 0.0000E+00 –2.0000E+06 –4.0000E+06 –6.0000E+06 –8.0000E+06 –1.0000E+07 –1.2000E+07 –1.4000E+07 –1.6000E+07 –1.8000E+07 –2.0000E+07 –2.0817E+07 (e) 10 m coal pillar Figure 3: Vertical stress distribution in coal pillars with diﬀerent widths and surrounding rock of roadway along goaf when the workface was advanced 50 m. Geoﬂuids 5 18 Vertical stress (MPa) 16 14 12 10 8 6 4 0 2 4 6 Coal pillar width (m) 8 6 m coal pillar 9 m coal pillar 7 m coal pillar 10 m coal pillar 10 8 m coal pillar Figure 4: Vertical stress distribution law of coal pillars with diﬀerent widths when the workface was advanced 50 m. goaf, 9106 workface, and section and mining area coal pillar. In the model, x direction is the workface tendency, Y direction is the workface trend, the trend length of 9106 workface (x direction) is 200 m, and the trend length of 9106 workface (Y direction) is 400 m. In the model, the dip angle of rock strata and coal seam is 0°, coal pillars with width of 50 m are set around the model, the excavation of 9106 workface was carried out from Y =50 m (the open-oﬀ cut position), and the excavation was stopped at Y =400 m, with a total excavation of 350 m forward. The bottom of the model constrains the vertical displacement, and the front, rear, left, and right sides constrain the horizontal displacement. The upper surface of the model is a free surface, and the overburden strata except the roof of coal seam are loaded on the upper surface of the model in the form of uniform load (8.2 Mpa load). Physical and mechanical parameters of roof and ﬂoor strata of coal seam and workface in the model are shown in Table 1. 4. Deformation and Failure Law of 9106 Roadway along Goaf without Supporting Condition In order to study the deformation and failure law of 9106 roadway along goaf and determine the reasonable width of coal pillar and support parameters, in the following we focus on the simulation study of the deformation and failure law of 6 m, 7 m, 8 m, 9 m, and 10 m width coal pillars and surrounding rock of roadway along goaf without support when 9106 workface advanced 50 m (initial weighting of 9106 workface) and 200 m (square of 9106 workface) from the open-oﬀ cut position, including the stress ﬁeld and plastic zone distribution of coal pillar and surrounding rock of roadway along goaf. 4.1. Deformation and Failure Law of Coal Pillars with Diﬀerent Widths when Workface Advanced 50 m 4.1.1. Distribution of Vertical Stress in Coal Pillars and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 50 m from the open-oﬀ cut position, the vertical stress distribution of 6 m, 7 m, 8 m, 9 m, and 10 m coal pillars and surrounding rock of roadway along goaf under no support condition is shown in Figure 3. It can be seen that the vertical stress distribution of coal pillars with diﬀerent widths is basically the same, the vertical stress concentration area of coal pillars is inclined “elliptic,” and the vertical stress in coal pillars increased ﬁrst and then decreased from the side of roadway to the side of 9102 goaf. The peak value of vertical stress of coal pillar is closely related to the width of coal pillar. When the width of coal pillar is 6 m, 7 m, 8 m, 9 m, and 10 m, the peak value of vertical stress of coal pillar is 15.14 Mpa, 16.51 Mpa, 17.32 Mpa, 16.75 Mpa, and 15.95 Mpa, respectively, as shown in Figure 4. When the width of coal pillar increased from 6 m to 7 m, the peak value of vertical stress in coal pillar increased by 9.04%, indicating that the bearing capacity of coal pillar increased signiﬁcantly at this time, and the stability of coal pillar and surrounding rock of roadway along goaf was good. When the width of coal pillar increased from 7 m to 8 m, the peak value of vertical stress in coal pillar increased by 4.90%, indicating that the bearing capacity of coal pillar continued to increase, but the increase was greatly reduced. At this time, the bearing capacity of coal pillar tended to limit. When the width of coal pillar continued to increase, the peak value 6 Geoﬂuids FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Zone Zone Plane: on Colorby: state-average Plane: on Colorby: state-average None None Shear-p Shear-n shear-p Shear-n shear-p Shear-n shear-p tension-p Shear-n shear-p tension-p Shear-p tension-p Shear-p Tension-n shear-p tension-p Shear-p tension-p Tension-n tension-p Tension-p Tension-p (a) 6 m coal pillar FLAC3D 5.00 (b) 7 m coal pillar FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Zone Zone Plane: on Colorby: state-average Plane: on Colorby: state-average None None Shear-n shear-p Shear-n shear-p Shear-n shear-p tension-p Shear-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-p Shear-p Shear-p tension-p Shear-p tension-p Tension-n shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-n tension-p Tension-p Tension-p (c) 8 m coal pillar (d) 9 m coal pillar FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Zone Plane: on Colorby: state-average None Shear-n shear-p Shear-n shear-p tension-p Shear-p Shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-p (e) 10 m coal pillar Figure 5: Plastic zone distribution in coal pillars with diﬀerent widths and surrounding rock along goaf when the workface was advanced 50 m. Geoﬂuids 7 FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Plane: on Calculated by: volumetric averaging Contour of ZZ-stress Plane: on Calculated by: volumetric averaging 4.7289E+05 4.0350E+05 0.0000E+00 0.0000E+00 –2.5000E+06 –2.5000E+06 –5.0000E+06 –5.0000E+06 –7.5000E+06 –7.5000E+06 –1.0000E+07 –1.0000E+07 –1.2500E+07 –1.2500E+07 –1.5000E+07 –1.5000E+07 –1.7500E+07 –1.7500E+07 –2.0000E+07 –2.0000E+07 –2.2500E+07 –2.2500E+07 –2.5000E+07 –2.5000E+07 –2.7500E+07 –2.7500E+07 –3.0000E+07 –3.0483E+07 –2.9745E+07 (a) 6 m coal pillar (b) 7 m coal pillar FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Contour of ZZ-stress Plane: on Calculated by: volumetric averaging Plane: on Calculated by: volumetric averaging 4.8958E+05 5.4109E+05 0.0000E+00 0.0000E+00 –2.5000E+06 –2.5000E+06 –5.0000E+06 –5.0000E+06 –7.5000E+06 –7.5000E+06 –1.0000E+07 –1.2500E+07 –1.0000E+07 –1.5000E+07 –1.2500E+07 –1.7500E+07 –1.5000E+07 –2.0000E+07 –1.7500E+07 –2.2500E+07 –2.0000E+07 –2.5000E+07 –2.2500E+07 –2.7500E+07 –2.5000E+07 –2.8761E+07 –2.7372E+07 (c) 8 m coal pillar (d) 9 m coal pillar FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Plane: on Calculated by: volumetric averaging 5.6174E+05 0.0000E+00 –2.5000E+06 –5.0000E+06 –7.5000E+06 –1.0000E+07 –1.2500E+07 –1.5000E+07 –1.7500E+07 –2.0000E+07 –2.2500E+07 –2.5000E+07 –2.6220E+07 (e) 10 m coal pillar Figure 6: Vertical stress distribution in coal pillars with diﬀerent widths and surrounding rock of roadway along goaf when the workface was advanced 200 m. 8 Geoﬂuids Vertical stress (MPa) 25 20 15 10 5 0 0 2 4 6 Coal pillar width (m) 8 6 m coal pillar 9 m coal pillar 7 m coal pillar 10 m coal pillar 10 8 m coal pillar Figure 7: Vertical stress distribution law of coal pillars with diﬀerent widths when the workface was advanced 200 m. of vertical stress in coal pillar showed a downward trend, indicating that the width of 7 m and 8 m was the critical width for the change of coal pillar bearing capacity. In conclusion, considering the recovery rate of coal resources, reasonable coal pillar width should be 7 m and 8 m. 4.1.2. Plastic Zone Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 50 m from the open-oﬀ cut position, the plastic zone in 6 m, 7 m, 8 m, 9 m, and 10 m coal pillars and surrounding rock of roadway along goaf were distributed without supporting condition, as shown in Figure 5. It can be seen that with the increase of coal pillar width, the roof and ﬂoor failure area of coal pillar and roadway along goaf have gradually decreased. When the width of the coal pillar is 6 m, the coal pillar presented a plastic failure state as a whole, and the roof and ﬂoor of roadway along goaf also appeared a certain range of plastic failure due to the mining inﬂuence of 9102 goaf and 9106 workface mining. When the width of coal pillar was 7 m, the overall bearing capacity of coal pillar increased, some elastic zones appeared in coal pillar, and the plastic failure zone of roof and ﬂoor of roadway decreased. When the width of coal pillar was 8 m, with the continuous increase of the bearing capacity of coal pillar, the elastic zone range of coal pillar and roof and ﬂoor of roadway along goaf also further increased, and there was a certain degree of connectivity. When the width of coal pillar was 9 m and 10 m, the plastic failure range of coal pillar continues to decrease, only the plastic failure zone appeared to a certain extent on both sides of coal pillar, and the elastic zone range of coal pillar and roof and ﬂoor of roadway along goaf continued to increase and connect. 4.2. Deformation and Failure Law of Coal Pillars with Diﬀerent Widths when Workface Advanced 200 m 4.2.1. Vertical Stress Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 200 m from the open-oﬀ cut position, the vertical stress distribution of 6 m, 7 m, 8 m, 9 m, and 10 m coal pillars and surrounding rock of roadway along goaf without supporting condition are shown in Figure 6. It can be seen that the vertical stress distribution of coal pillars with diﬀerent widths and surrounding rock of roadway along goaf is basically the same. With the increase of coal pillar width, the vertical stress of coal pillar and surrounding rock of roadway along goaf increased ﬁrst and then decreased, and as the width of coal pillar increased, the concentration range of vertical stress of coal pillar increased. When the width of coal pillar is 6, 7, 8, 9, and 10 m, the peak values of vertical stress are 16.57 Mpa, 21.96 Mpa, 23.67 Mpa, 23.71 Mpa, and 23.12 Mpa, respectively, as shown in Figure 7. The results showed that the peak value of vertical stress increased ﬁrstly and then decreased slowly with the increase of pillar width. When the width of coal pillar increased from 6 m to 7 m, the peak value of vertical stress of coal pillar increased by 32.5%, indicating that the bearing capacity of coal pillar was greatly improved. When the width of coal pillar increased from 7 m to 8 m, the peak value of vertical stress of coal pillar increased by 7.7%, indicating that the bearing capacity of coal pillar continued to increase, but the increase amplitude decreased. At this time, the bearing capacity of coal pillar had approached the limit. When the width of coal pillar increased from 8 m to 9 m, the peak value of vertical stress increased by 0.16%. When the width of coal pillar increased from 9 m to 10 m, the peak value of vertical Geoﬂuids 9 FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting goup, Inc. Zone Zone Plane: on Colorby: state-average Plane: on Colorby: state-average None None shear-n shear-p Shear-n shear-p Shear-n shear-p tension-p Shear-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-p Shear-p Shear-p tension-p Shear-p tension-p Tension-n shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-n tension-p Tension-p Tension-p (a) 6 m coal pillar (b) 7 m coal pillar FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Zone Plane: on Colorby: state-average Zone Plane: on Colorby: state-average None None Shear-n shear-p Shear-n shear-p Shear-n shear-p tension-p Shear-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-p Shear-p Shear-p tension-p Shear-p tension-p Tension-n shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-n tension-p Tension-p Tension-p (c) 8 m coal pillar (d) 9 m coal pillar FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Zone Plane: on Colorby: state-average None Shear-n shear-p Shear-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-p Shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-p (e) 10 m coal pillar Figure 8: Plastic zone distribution in coal pillars with diﬀerent widths and surrounding rock of roadway along goaf when the workface was advanced 200 m. stress of coal pillar decreased by 2.48%, indicating that the coal pillar had reached the yield limit state and the plastic zone range of coal pillar further increased. 4.2.2. Plastic Zone Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 200 m from the open-oﬀ cut position, the plastic zone in 6 m, 7 m, 8 m, 9 m, and 10 m coal pillars and surrounding rock of roadway along goaf were distributed without supporting condition, as shown in Figure 8. It can be seen that the 9102 goaf had a great inﬂuence on the deformation and failure of surrounding rock of roadway along goaf, which made the plastic failure zone formed after mining of 9102 and 9106 workface connect with the plastic failure zone of surrounding rock of roadway along goaf. When the width of the coal pillar was 6 m, the overall coal pillar was in a plastic failure state, and the roof and ﬂoor of roadway along goaf were in a plastic failure state in a certain range, indicating that the mining inﬂuence of 9102 goaf and 9106 workface mining was large, resulting in large stress of the coal pillar, and the bearing capacity of the coal pillar was weak. When the width of coal pillar was 7 m, some 10 Geoﬂuids (a) Bolt (cable) supporting section (b) Anchor (cable) overall support Figure 9: Anchor (cable) support model. FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Cable Cable Colorby: Uniform Colorby: Uniform Uniform Uniform Contour of ZZ-stress Contour of ZZ-stress Calculated by: volumetric averaging Calculated by: volumetric averaging 6.5392E+05 6.2881E+05 0.0000E+00 0.0000E+00 –2.5000E+06 –2.5000E+06 –5.0000E+06 –5.0000E+06 –7.5000E+06 –7.5000E+06 –1.0000E+07 –1.0000E+07 –1.2500E+07 –1.2500E+07 –1.5000E+07 –1.5000E+07 –1.7500E+07 –1.7500E+07 –2.0000E+07 –2.0000E+07 –2.2500E+07 –2.2500E+07 –2.4801E+07 –2.4916E+07 (a) 800 mm row distance (b) 900 mm row distance FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Cable Colorby: Uniform Uniform Contour of ZZ-stress Calculated by: volumetric averaging 6.2910E+05 0.0000E+00 –2.0000E+06 –4.0000E+06 –6.0000E+06 –8.0000E+06 –1.0000E+07 –1.2000E+07 –1.4000E+07 –1.6000E+07 –1.8000E+07 –2.0000E+07 –2.0792E+07 (c) 1000 mm row distance Figure 10: Vertical stress distribution in 7 m width coal pillar and surrounding rock of roadway along goaf under the condition of diﬀerent bolt (cable) support row distance. Geoﬂuids 11 22 20 Vertical stress (MPa) 18 16 14 12 10 8 6 4 –1 0 1 2 3 4 5 Coal pillar width (m) 6 7 8 Array pitch 800 mm Array pitch 900 mm Array pitch 1000 mm Figure 11: Vertical stress distribution law in 7 m width coal pillar under the condition of diﬀerent bolt (cable) support row distance. elastic zones appeared in the roof and ﬂoor of coal pillar and roadway along goaf, indicating that the bearing capacity of coal pillar was improving to some extent at this time. When the width of coal pillar was 8 m, the elastic zone of coal pillar and roof and ﬂoor of roadway along goaf expanded and connected with the elastic zone of solid coal side, indicating that the bearing capacity of coal pillar continued to increase and can eﬀectively withstand the inﬂuence of mining in 9102 goaf and 9106 workface mining. When the widths of the coal pillar were 9 m and 10 m, the elastic zone of the roof and ﬂoor of coal pillar and roadway along goaf was further expanded, and the coal pillar presented a state of plastic zone on both sides and elastic zone in the middle, indicating that the coal pillar can fully withstand the mining inﬂuence of 9102 goaf and 9106 workface mining at this time. In summary, the width of coal pillar should be greater than or equal to 8 m. Considering the recovery rate of coal resources, a reasonable width of coal pillar should be 8 m. 5. Deformation and Failure Law of 9106 Roadway along Goaf under Supporting Condition Deformation and failure law of 9106 roadway along goaf without supporting condition showed that the 9106 roadway along goaf was greatly aﬀected by 9102 goaf and 9106 workface mining. Especially when 9106 workface advanced 200 m from the open-oﬀ cut position (square of 9106 workface), the vertical stress of coal pillar and surrounding rock of roadway along goaf was generally greater than that when 9106 workface advanced 50 m from the open-oﬀ cut position (initial weighting of 9106 workface). At the same time, the simulation study also showed that the deformation and failure law of coal pillar and surrounding rock of roadway along goaf were closely related to the width of coal pillar. Under the premise of considering safe production and improving the recovery rate of coal resources, 8 m width coal pillar should be preferred, followed by 7 m width coal pillar. Therefore, the following simulation study focused on the deformation and failure law of 7 m and 8 m width coal pillar and surrounding rock of roadway along goaf under diﬀerent support conditions when 9106 workface advanced 200 m from the open-oﬀ cut position, including the stress ﬁeld and plastic zone distribution in coal pillar and surrounding rock of roadway along goaf, to determine the best coal pillar width and support parameters. Using FLAC3D numerical simulation software to establish the supporting model of coal pillar and roadway along goaf under diﬀerent supporting conditions and analyze the deformation and failure law of coal pillar and surrounding rock of roadway along goaf. As shown in Figure 9, 9106 roadway along goaf adopted bolt-anchor cable combined support (anchor cable only arranged in the roof of roadway). The concrete supporting parameters are ﬁxed anchor rod distance is 1000 mm, and row distance is 800 mm, 900 mm, and 1000 mm. At the same time, the ﬁxed anchor rope distance is 2000 mm, and when the anchor rod distance is 800 mm, the anchor row distance is 1600 mm; and when the anchor rod distance is 900 mm, the anchor rope distance is 1800 mm; and when the anchor rod distance is 1000 mm, the anchor rope distance is 2000 mm. 5.1. Deformation and Failure Law of 7 m Width Coal Pillar and Surrounding Rock of Roadway along Goaf 5.1.1. Vertical Stress Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 200 m from the open-oﬀ cut position, under the condition of the row distance of bolt support was 800 mm, 900 mm, and 1000 mm (the support spacing is 1000 mm), the vertical stress distribution of 7 m width coal pillar and surrounding rock of roadway along goaf is shown in Figure 10. It can be seen that the vertical stress distribution of 7 m width coal pillar and surrounding rock of roadway along goaf was roughly the same under the conditions of 800 mm, 900 mm, and 1000 mm bolt support row distance. Under the inﬂuence of 9102 gob and 9106 workface mining, “elliptical” stress concentration occurred in coal pillars and solid coal, and the stress concentration range on the side of solid coal was larger. The vertical stress distribution in coal pillar from the roadway to the 9102 goaf showed a trend of increasing ﬁrst and then decreasing, and the vertical stress in coal pillar on the side of roadway was signiﬁcantly greater than that in the coal pillar of 9102 goaf, as shown in Figure 11. The vertical stress distribution in coal pillar showed that the bolt (cable) support eﬀect was good, and the plastic zone of surrounding rock of roadway side decreased and the supporting capacity increased. In addition, the peak value of vertical stress in coal pillar decreased with the increase of bolt (cable) support row distance, indicating that reducing bolt (cable) support row distance can increase the supporting capacity of coal pillar and surrounding rock of roadway along goaf to a certain extent, and reducing the inﬂuence 12 Geoﬂuids FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Zone Zone Plane: on Colorby: state-average Plane: on Colorby: state-average None None Shear-n Shear-n Shear-n shear-p Shear-n shear-p Shear-n shear-p tension-p Shear-n shear-p tension-p Shear-n tension-n Shear-n tension-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-p Shear-p Shear-p tension-p Shear-p tension-p Tension-n Tension-n Tension-n shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-n tension-p Tension-p Tension-p (a) 800 mm row distance (b) 900 mm row distance FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Zone Plane: on Colorby: state-average None Shear-p Shear-n Shear-n shear-p Shear-n shear-p tension-p Shear-n tension-n shear-p tension-p Shear-p tension-p Tension-n Tension-n shear-p tension-p Tension-n tension-p Tension-p (c) 1000 mm row distance Figure 12: Plastic zone distribution of 7 m width coal pillars and surrounding rock of roadway along goaf under the condition of diﬀerent bolt (cable) support row distance. of 9102 goaf and 9106 workface mining on coal pillar and surrounding rock of roadway. 5.1.2. Plastic Zone Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 200 m from the open-oﬀ cut position, under the condition of the row distance of bolt support was 800 mm, 900 mm, and 1000 mm (the support spacing was 1000 mm), the plastic zone distribution of 8 m width coal pillar and surrounding rock of roadway along goaf is shown in Figure 12. It can be seen that under the conditions of bolt support row distance of 800 mm, 900 mm, and 1000 mm, the coal pillar and surrounding rock of roadway along goaf occurred plastic failure to varying degrees due to the inﬂuence of 9102 goaf and 9106 workface mining. With the increase of bolt (cable) support row distance, the plastic failure zone of coal pillar and surrounding rock increased accordingly. When the bolt row distance was 800 mm, most of the coal pillar was elastic zone, indicating that the bolt row distance of 800 mm can eﬀectively increase the supporting capacity of coal pillar and surrounding rock of roadway along goaf. When the bolting row distance was 900 mm and 1000 mm, the failure zone of coal pillar and surrounding rock of roadway along goaf increased. In sum- mary, when a 7 m width coal pillar was left, the 800 mm bolt support row distance can eﬀectively reduce the impact of 9102 goaf and 9106 workface mining and increase the supporting capacity of coal pillar. 5.2. Deformation and Failure Law of 8 m Width Coal Pillar and Surrounding Rock of Roadway along Goaf 5.2.1. Vertical Stress Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 200 m from the open-oﬀ cut position, under the condition of the row distance of bolt support was 800 mm, 900 mm, and 1000 mm (the support spacing was 1000 mm), the vertical stress distribution of 8 m width coal pillar and surrounding rock of roadway along goaf is shown in Figure 13. It can be seen that the vertical stress distribution in 8 m width coal pillar and surrounding rock of roadway along goaf was roughly the same under the conditions of 800 mm, 900 mm, and 1000 mm bolt support row distance. The vertical stress distribution in coal pillar showed a trend of increasing ﬁrst and then decreasing, and the vertical stress in coal pillar on the side of roadway was signiﬁcantly greater than that in the coal pillar of 9102 goaf, indicating Geoﬂuids 13 FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Contour of ZZ-stress Plane: on Calculated by: volumetric averaging Plane: on Calculated by: volumetric averaging 6.4944E+05 6.4270E+05 0.0000E+00 0.0000E+00 –2.0000E+06 –2.5000E+06 –4.0000E+06 –5.0000E+06 –6.0000E+06 –7.5000E+06 –8.0000E+06 –1.0000E+07 –1.0000E+07 –1.2500E+07 –1.2000E+07 –1.4000E+07 –1.5000E+07 –1.6000E+07 –1.7500E+07 –1.8000E+07 –2.0000E+07 –2.0000E+07 –2.2500E+07 –2.0612E+07 –2.4650E+07 (a) 800 mm row distance (b) 900 mm row distance FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Contour of ZZ-stress Plane: on Calculated by: volumetric averaging 6.4353E+05 0.0000E+00 –2.0000E+06 –4.0000E+06 –6.0000E+06 –8.0000E+06 –1.0000E+07 –1.2000E+07 –1.4000E+07 –1.6000E+07 –1.8000E+07 –2.0000E+07 –2.0146E+07 (c) 1000 mm row distance Figure 13: Vertical stress distribution in 8 m width coal pillar and surrounding rock of roadway along goaf under the condition of diﬀerent bolt (cable) support row distance. that the supporting eﬀect was good, as shown in Figure 14. When the bolting row distance was 800 mm and 900 mm, the stress concentration range in coal pillar on the side of roadway was large. On the contrary, when the bolt supporting row distance was 1000 mm, the stress concentration range was small. The results showed that when the bolting row distance was 800 mm and 900 mm, the integrity of coal pillar was better, the failure area of coal pillar was smaller, and the supporting capacity was larger. However, when the bolting row distance was 1000 mm, the condition was opposite. 25 Vertical stress (MPa) 20 15 10 5 0 0 2 4 6 Coal pillar width (m) 8 Array pitch 800 mm Array pitch 900 mm Array pitch 1000 mm Figure 14: Vertical stress distribution law in 8 m width coal pillar under the condition of diﬀerent bolt (cable) support row distance. 5.2.2. Plastic Zone Distribution in Coal Pillar and Surrounding Rock of Roadway along Goaf. When the 9106 workface advanced 200 m from the open-oﬀ cut position, under the condition of the row distance of bolt support was 800 mm, 900 mm, and 1000 mm (the support spacing was 1000 mm), the plastic zone distribution of 8 m width coal pillar and surrounding rock of roadway along goaf is shown in Figure 15. It can be seen that with the increase of bolt (cable) support row distance, the plastic failure zone of coal pillar and surrounding rock increased accordingly. When the bolting row distance was 800 mm and 900 mm, the damage range in coal pillar was small. When the bolting 14 Geoﬂuids FLAC3D 5.00 FLAC3D 5.00 © 2012 Itasca consulting group, Inc. © 2012 Itasca consulting group, Inc. Zone Zone Plane: on Colorby: state-average Plane: on Colorby: state-average None None Shear-n shear-p Shear-n Shear-n shear-p tension-p Shear-n shear-p Shear-n shear-p tension-p Shear-p Shear-p Shear-p tension-p Shear-p tension-p Tension-n shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-n tension-p Tension-p Tension-p (a) 800 mm row distance (b) 900 mm row distance FLAC3D 5.00 © 2012 Itasca consulting group, Inc. Zone Plane: on Colorby: state-average None Shear-n Shear-n shear-p Shear-n shear-p tension-p Shear-p Shear-p tension-p Tension-n shear-p tension-p Tension-n tension-p Tension-p (c) 1000 mm row distance Figure 15: Plastic zone distribution of 8 m width coal pillars and surrounding rock of roadway along goaf under the condition of diﬀerent bolt (cable) support row distance. row distance was 1000 mm, the damage range in coal pillar increased. In summary, when setting 8 m width coal pillar, the 800 mm bolt support row distance can eﬀectively reduce the impact of 9102 goaf and 9106 workface mining and increase the supporting capacity of coal pillar. 6. Conclusions Based on hydrogeological data of 9106 workface of Hengsheng Coal Industry, a mining numerical calculation model of 9106 workface was established by using FLAC numerical simulation software. The deformation and failure laws in 6 m, 7 m, 8 m, 9 m, and 10 m width coal pillars and surrounding rock of roadway along goaf without support were simulated and analyzed, as well as the deformation and failure laws in 7 m and 8 m width coal pillars and surrounding rock of roadway along goaf under diﬀerent support conditions. The main conclusions are as follows: (1) Vertical stress distribution of coal pillars with diﬀerent widths is basically the same. The vertical stress in coal pillars increases ﬁrstly and then decreases from the side of roadway to the side of 9102 goaf, and the vertical stress of coal pillars at the side of roadway is obviously greater than that at 9102 goaf (2) With the increase of the width of coal pillar, the failure area of coal pillar and roof and ﬂoor of roadway along goaf decreases gradually, the elastic area increases gradually, and the bearing capacity increases gradually (3) Reducing the bolt (cable) support row distance can reduce the plastic zone of coal pillar and surrounding rock of roadway along goaf to a certain extent and increase the bearing capacity of coal pillar and surrounding rock of roadway along goaf, so as to reduce the inﬂuence of 9102 goaf and 9106 workface mining on coal pillar and surrounding rock of roadway along goaf (4) When 7 m coal pillar is left in 9106 roadway along goaf, the optimal bolt row distance is 800 mm; when 8 m coal pillar is left, the optimal bolt row distance is 900 mm, which can not only ensure the stability of roadway surrounding rock in the mining process of 9106 workface but also produce more coal resources and save support cost Data Availability The authors declare that they have no conﬂicts of interest regarding the publication of this paper. Geoﬂuids Conflicts of Interest The authors declare that they have no conﬂicts of interest regarding the publication of this paper. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 51974010). References [1] Z. Kexue, “Determination of reasonable width of roadway pillar in gob driving roadway of deep coal seam group,” Journal of China Coal Society, vol. 36, no. S1, pp. 28–35, 2011. [2] B. Jianbiao, H. Chaojiong, and H. Hanfu, “Numerical simulation study on stability of narrow coal pillar in gob driving roadway,” Chinese Journal of Rock Mechanics and Engineering, vol. 23, no. 20, pp. 3475–3479, 2004. [3] L. Zhenlei, D. Linming, W. Cai et al., “Study on rock burst mechanism of fault pillar in deep thick coal seam,” Chinese Journal of Rock Mechanics and Engineering, vol. 32, no. 2, pp. 333–342, 2013. [4] X. Zheng, Y. Zhigang, and Z. 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https://openalex.org/W2901264857	https://europepmc.org/articles/pmc6294631?pdf=render	English	null	The association of auditory integration training in children with autism spectrum disorders among Chinese: a meta-analysis	Bioscience reports	2,018	cc-by	6,550	Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Randomized controlled trials (RCTs) have reported an inconsistent relationship about the au- ditory integration training (AIT) in children with autism spectrum disorders (ASD) among Chi- nese. The current study was to investigate the efficacy of AIT for children with ASD compared with those in control group by using meta-analysis. Relevant trials published were identified by an electronic search of PubMed, CENTRAL, EMBASE, WanFang, CNKI, and SinoMed databases up to December 31, 2017. Outcome of interest included childhood autism rat- ing scale (CARS), autism behavior checklist (ABC), intelligence quotient (IQ), and autism treatment evaluation checklist (ATEC). Standardized mean difference (SMD) with 95% con- fidence intervals (CIs) was calculated using a random-effect model. Thirteen RCTs with 976 children with ASD were included for analysis. The pooled SMD showed that children with ASD had significantly lower ABC scores [summary SMD = −0.58, 95%CI = −0.79 to −0.38] and ATEC scores [summary SMD = −0.75, 95%CI = −1.05 to −0.45] in AIT group compared with that in control group. The analysis of pooled statistics put forward AIT could increase the IQ score when compared with that in control group [summary SMD = 0.59, 95%CI = 0.41–0.77]. A negative association was found about CARS scores between AIT group and control group. No publication bias was found and no single study had essential effect on the pooled results. In conclusions, AIT can reduce the score of ABC and ATEC and can increase the IQ score among children with ASD in Chinese. Therefore, it is recommended for Chinese children with ASD to receive AIT. ⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution cense 4 0 (CC BY) c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). The association of auditory integration training in children with autism spectrum disorders among Chinese: a meta-analysis 1Department of Pediatric Health Care, Jinan Children’s Hospital of Shandong University, Jinan, Shandong 250022, China; 2Department of Pediatrics, Shandong Provincial Hospital Afﬁliated to Shandong University, Jinan, Shandong 250022, China C d Zhongtao Gai (gai hongtaojn@163 com) Received: 15 August 2018 Revised: 05 November 2018 Accepted: 06 November 2018 Received: 15 August 2018 Revised: 05 November 2018 Accepted: 06 November 2018 Accepted Manuscript Online: 14 November 2018 Version of Record published: 11 December 2018 access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Introduction Autism spectrum disorders (ASD) or autism refers to a wide range of related cognitive and behavioral dis- orders [1]. ASD are characterized by social and communication difficulties, alongside repetitive behaviors and special interests [2]. Data from previous publication [3] indicated that every 88 children may have one with ASD, it developed slowly and the cause of ASD cannot be completely determined. It also showed that approximately 50% of children with ASD had sensitive hearing phenomena; moreover, paranoid behav- ior and poor verbal were closely linked to auditory abnormalities [4]. However, therapies were developed to overcome the common auditory sensitivity changes in autistic patients and collectively referred to as auditory integration therapies. Auditory integration training (AIT), which was first developed in France in 1982 by Berard [5], was one of the therapies. AIT involves 10 h of listening to electronically modified music delivered by headphones during two half-hour sessions each day for 10 days. The AIT device uses filtering to dampen the peak frequencies to which the individual is ‘hypersensitive’ and delivers sounds modulated by random dampening of high and low frequencies and intensities [6]. y g g Sinha et al. conducted three reviews and meta-analysis to assess the association of AIT and other sound therapies for adults or children with ASD [7–9]. However, in those results, they concluded that there 1 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 is not sufficient evidence to support AIT use. At present, there are more and more related RCTs about AIT treatment for children with ASD in China. Nonetheless, for the treatment of AIT, there existed marked disparities among studies owing to the variable research designs and limited sample sizes. Accordingly, we performed a meta-analysis to estimate the effect of AIT treatment for children with ASD in Chinese. is not sufficient evidence to support AIT use. At present, there are more and more related RCTs about AIT treatment for children with ASD in China. Nonetheless, for the treatment of AIT, there existed marked disparities among studies owing to the variable research designs and limited sample sizes. Accordingly, we performed a meta-analysis to estimate the effect of AIT treatment for children with ASD in Chinese. Data extraction Two investigators screened the titles and abstracts of potentially relevant studies. The same two reviewers retrieved the full text of relevant studies for further review. A third senior investigator resolved any discrepancies between reviewers. If reviewers suspected an overlap of populations in a report, they contacted the corresponding author for clarification; we excluded studies with a clear overlap. The same pair of reviewers extracted study details independently. A third investigator reviewed all data entries. We extracted the following data: author, publications years, mean age or age range, diagnostic criteria, treatment method both for AIT group and control group, sample size, outcomes of interest and scores (mean +−SD) for each outcome. Meanwhile, trial validity assessment was done independently, and a trial quality assessment as assigned (A to C) according to the Cochrane Reviewers’ Handbook 4.2.2 [13]. Method and materials Study design We performed the present meta-analysis adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statements [10]. Identiﬁcation and selection of studies We conducted a broad search of four databases, including PubMed, CENTRAL, EMBASE, WanFang, CNKI, and SinoMed databases, to identify relevant studies up to December 31, 2017. The following Mesh terms were used: “auditory integration training” OR “auditory therapy” AND “autism” OR “autistic children” OR “autism spectrum disorder” AND “Chinese”. Additional references were searched through manual searches of the reference lists and specialist journals. No language restrictions were applied. To be eligible for inclusion in our study, publications had to meet all the following criteria: (1) study conducted with randomized controlled trials (RCTs); (2) reported the studies on Chinese children; (3) children with ASD are diagnosed using diagnostic manual of mental disorders (4th edn) (DSM-IV) [11] and international classification of diseases-10th (ICD-10) [12] or diagnosed using a standard diagnostic instrument; (4) AIT group was accepted additionally with AIT based on the treatment of the control group; (5) patients with ASD were Chinese children only; (6) reported outcomes of interest (i.e. childhood autism rating scale (CARS), autism behavior checklist (ABC), intelligence quotient (IQ), and autism treatment evaluation checklist (ATEC)); (7) availability of mean and standard deviation (SD) of scores about CARS, ABC, IQ, or ATEC. Furthermore, children with impaired brain development, children with epilepsy and mental disorders, hyperemia and inflammation of the middle ear, deafness, and other abnormal hearing were excluded. c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Study characteristics The flowchart of Figure 1 revealed detailed screening process of our analysis. In terms of the initial searching strategy, a total of 321 articles were obtained preliminarily. Additional 2 articles were identified through other sources. A total of 120 articles were removed after duplicated in different databases. After screening based on titles and abstracts, 160 articles were excluded. Subsequently, the full texts of remaining articles were carefully reviewed, and 30 articles of them were ruled out for the following reasons: 3 articles were duplicate publications; 10 articles failed to provide mean or SD of scores for outcomes; 10 articles were reviews; the other 7 articles had no suitable outcomes. Eventually, 13 RCTs [21–33] involving 489 children with ASD in AIT group and 487 children with ASD in control group were included in our meta-analysis. The main characteristics of the 13 RCTs are listed in Table 1. c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Statistical analysis The relationship about AIT in children with ASD was pooled using standardized mean difference (SMD), which could control heterogeneity between different studies and some other influencing factors, with 95% confidence intervals (CIs) for CARS, ABC, IQ, or ATEC [14]. A random-effects model for the current meta-analysis was used. Subgroup analysis by diagnostic criteria was performed. Heterogeneity of pooled results was assessed using Cochran’s Q-test and the Higgins I2 statistic [15]. Pheterogeneity < 0.1 or I2 > 50% suggested significant heterogeneity among the included studies [16]. Meta-regression was used to assess the potential of important covariates to exert substantial impact on between-study heterogeneity [17]. Begg’s funnel plot [18] and Egger’s linear regression test [19] were conducted to verify publication bias, and a value of P<0.05 was considered statistically significant. A sensitivity analysis [20] by exclusion of one study at the time was performed to assess the stability of results and potential sources of heterogeneity. All statistical analyses were conducted using the STATA software, version 12.0 (STATA Corporation, College Station, TX, U.S.A.). Unless otherwise specified, all P values were two-sided. 2 c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Figure 1. Selection process for clinical trials included in the meta-analysis Figure 1. Selection process for clinical trials included in the meta-analysis Main result of AIT for the effect of ABC There were 10 articles [21–23,25–31] included to assess the association about AIT for the effect of ABC among chil- dren with ASD. With significant heterogeneity (I2 = 43.3%, Pheterogeneity = 0.070), the analysis of primary pooled statistics put forward that children with ASD had significantly lower ABC scores in AIT group compared with that in control group [summary SMD = −0.58, 95%CI = −0.79 to −0.38] (Figure 2). Among the 10 articles, 9 of which were using DSM-IV diagnostic criteria, and only 1 article diagnosed ASD using ICD-10. Significant relationship was also found in the subgroup of using DSM-IV diagnostic criteria [summary SMD = −0.58, 95%CI = −0.81 to −0.35, I2 = 49.2%, Pheterogeneity = 0.046]. g y As availability of significant heterogeneity among pooled results, we then performed univariate meta-regression to explore whether the reason of heterogeneity was associated with covariates of publication year, case numbers and 3 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Table 1 Characteristics of the included studies about auditory integration training in children with autism spectrum disorders among Chinese Study, year [reference number] Diagnostic criteria Age (range or Mean +− SD) OutcomeQuality AIT group Control group n Treatment method Mean +−SD (scores) n Treatment method Mean +−SD (scores) Liu, Y.M. et al., 2015 [19] DSM-IV 5.15 +−0.94 CARS A 20 Auditory integration training and individualized training 34.03 +−4.64 25 No training 38.96 +−3.95 Liu, Y.M. et al., 2015 [19] DSM-IV 5.15 +−0.94 ABC A 20 Auditory integration training and individualized training 85.45 +−8.01 25 No training 102.84 +− 20.97 Liu,Y.M. et al., 2015 [19] DSM-IV 5.15 +−0.94 IQ A 20 Auditory integration training and individualized training 61.15 +−16.89 25 No training 47.86 +−13.52 Sun, Y.Y. et al., 2014 [20] DSM-IV 3–9 ABC B 22 Auditory integration training 73.77 +−17.91 21 Music therapy 88.2 +−18.37 Yu, D.M. et al., 2016 [21] DSM-IV 3–7 ABC A 40 Routine rehabilitation training and auditory integration training 84.1 +−11.6 40 Routine rehabilitation training 86.5 +−11.6 Wei, B.H. et al., 2012 [22] CCMD-3 4–6 ATEC B 43 Guided education training and auditory integration training 71.73 +−8.49 43 Guided education training 78.69 +−10.02 Xie, J.N. et al., 2014 [23] ICD-10 2.5–5.5 ABC B 47 Auditory integration training 50.89 +−21.09 39 No training 67.03 +−29.25 Wang, Y.J. Continued over c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Main result of AIT for the effect of ABC et al., 2016 [24] DSM-IV 4.07 +−1.54 ABC B 35 Routine rehabilitation training and auditory integration training 94.6 +−13.17 35 Routine rehabilitation training 94.26 +−10.91 Wang, Y.J. et al., 2016 [24] DSM-IV 4.07 +−1.54 CARS B 35 Routine rehabilitation training and auditory integration training 38.66 +−6.33 35 Routine rehabilitation training 39.57 +−9.19 Wang, Y.J. et al., 2016 [24] DSM-IV 4.07 +−1.54 IQ B 35 Routine rehabilitation training and auditory integration training 60.89 +−16.52 35 Routine rehabilitation training 56.52 +−17.23 Li, W.J. et al., 2013 [25] DSM-IV 3–6 ABC B 24 Routine rehabilitation training and auditory integration training 57.09 +−11.72 26 Routine rehabilitation training 63.52 +−10.1 Li, W.J. et al., 2013 [25] DSM-IV 3–6 IQ B 24 Routine rehabilitation training and auditory integration training 69.58 +−12.39 26 Routine rehabilitation training 64.63 +−12.01 Zhang, Y.H. et al., 2013 [19] DSM-IV 5.6 +−2.1 ABC B 43 Comprehensive treatment and auditory integration training 68.3 +−9.1 43 Comprehensive treatment 75.1 +−11.6 Zhang, Y.H. et al., 2013 [26] DSM-IV 5.6 +−2.1 IQ B 43 Comprehensive treatment and auditory integration training 67.1 +−12.7 43 Comprehensive treatment 58.1 +−14.2 Wu, Y.Z. et al., 2016 [27] DSM-IV 3–9 ABC B 45 Comprehensive treatment and auditory integration training 64.37 +−9.25 45 Comprehensive treatment 73.68 +−11.26 Continued over Table 1 Characteristics of the included studies about auditory integration training in children with autism spectrum disorders among Chinese cs of the included studies about auditory integration training in children with autism spectrum nese Continued over 4 4 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Table 1 Characteristics of the included studies about auditory integration training in children with autism spectrum disorders among Chinese (Continued) Study, year [reference number] Diagnostic criteria Age (range or Mean +− SD) OutcomeQuality AIT group Control group n Treatment method Mean +−SD (scores) n Treatment method Mean +−SD (scores) Wu, Y.Z. et al., 2016 [27] DSM-IV 3–9 IQ B 45 Comprehensive treatment and auditory integration training 69.43 +−11.65 45 Comprehensive treatment 61.53 +−12.17 Zhang, J. et al., 2014 [28] DSM-IV 3–6 ABC A 40 Routine rehabilitation training and auditory integration training 80.3 +−10.26 40 Routine rehabilitation training 84.57 +−11.36 Wang, J.H. et al., 2017 [29] DSM-IV 2–8 ABC B 32 Routine rehabilitation training and auditory integration training 64.59 +−9.48 32 Routine rehabilitation training 74.14 +−12.84 Wang, J.H. Main result of AIT for the effect of ABC et al., 2017 [29] DSM-IV 2–8 IQ B 32 Routine rehabilitation training and auditory integration training 68.22 +−12.48 32 Routine rehabilitation training 59.12 +−13.29 Chen, W.H. et al., 2017 [30] CCMD-3 2–6 IQ B 48 Routine rehabilitation training and auditory integration training 67.42 +−12.98 48 Routine rehabilitation training 59.15 +−14.12 Ye, B. et al., 2014 [31] CCMD-3 5–7 ATEC B 50 Guided education training and auditory integration training 71.74 +−8.5 50 Guided education training 78.7 +−9.99 Abbreviations: ABC, autism behavior checklist; ATEC, autism treatment evaluation checklist; AIT, auditory integration training; CARS, childhood autism rating scale; CCMD-3, classiﬁcation of Chinese mental disorders and diagnostic criteria-3th; DSM-IV, diagnostic manual of mental disorders (4th edn); ICD-10, International classiﬁcation of diseases-10th; IQ, intelligence quotient; SD, standard deviation. cs of the included studies about auditory integration training in children with autism spectrum nese (Continued) Abbreviations: ABC, autism behavior checklist; ATEC, autism treatment evaluation checklist; AIT, auditory integration training; CARS, childhood autism rating scale; CCMD-3, classiﬁcation of Chinese mental disorders and diagnostic criteria-3th; DSM-IV, diagnostic manual of mental disorders (4th edn); ICD-10, International classiﬁcation of diseases-10th; IQ, intelligence quotient; SD, standard deviation. different diagnostic criteria. No significant contributions to between-study heterogeneity were found in this analysis (P = 0.341, 0.179, 0.521 for publication year, case numbers and different diagnostic criteria respectively). different diagnostic criteria. No significant contributions to between-study heterogeneity were found in this analysis (P = 0.341, 0.179, 0.521 for publication year, case numbers and different diagnostic criteria respectively). To check the influence of each individual study involved in our meta-analysis on the pooled SMD for ABC scores, we removed studies in sequence. The results were not materially altered, suggesting that the pooled SMD were stable and robust. Publication bias was assessed for ABC scores of AIT for children with ASD by Begg’s funnel plot and Egger’s linear regression test. In our meta-analysis, Begg’s test (Pr > \|z\| = 0.858) and Egger’s test (P > \|t\| = 0.289) show no obvious evidence of publication bias. As demonstrated in Figure 4, the symmetric shape of funnel plot supported the conclusion too. Main result of AIT for the effect of CARS Two articles [21,26] were included to explore the association between AIT and the effect of CARS of children with ASD. The forest plot of analysis is illustrated in Figure 2. The scores of CARS were not significant in the AIT group when compared with the control group [summary SMD = −0.61, 95%CI = −1.63 to 0.41, I2 = 84.9%, Pheterogeneity = 0.010]. Main result of AIT for the effect of ATEC There are two studies [24,33] included to assess the scores of ATEC between AIT group and control group. As a result, the ATEC score was significantly lower in AIT group compared with those in control group [summary SMD = −0.75, 95%CI = −1.05 to −0.45, I2 = 0.0%, Pheterogeneity = 0.998] (Figure 2). 5 5 c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Figure 2. Meta-analysis of randomized controlled trials about ABC scores, CARS scores and ATEC scores for children with ASD among Chinese between AIT group and control group Figure 2. Meta-analysis of randomized controlled trials about ABC scores, CARS scores and ATEC scores for children with ASD among Chinese between AIT group and control group Figure 3. Forest plots of RCTs evaluating IQ scores for children with ASD among Chinese between AIT group and control group Figure 3. Forest plots of RCTs evaluating IQ scores for children with ASD among Chinese between AIT group and control group Figure 3. Forest plots of RCTs evaluating IQ scores for children with ASD among Chinese between AIT group and control group Figure 3. Forest plots of RCTs evaluating IQ scores for children with ASD among Chinese between AIT g group c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). 6 c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). c⃝2018 The Author(s). Main result of AIT for the effect of CARS This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Figure 4. Begg’s funnel plots for assessment of publication bias about AIT for ABC scores Figure 5. Begg’s funnel plots for assessment of publication bias about AIT for IQ scores Main result of AIT for the effect of IQ Seven RCTs [21,26–29,31,32] were available to determine the effects of treatment on IQ in AIT group. The pooled result showed that the IQ score was significantly higher in AIT group than that in control group [summary SMD = 0.59, 95%CI = 0.41–0.77, I2 = 0.0%, Pheterogeneity = 0.729]. Figure 3 showed the forest plot of SMD with corresponding 95%CI about IQ scores between AIT group and control group. Six of the 7 RCTs were diagnosed by DSM-IV, and only Figure 4. Begg’s funnel plots for assessment of publication bias about AIT for ABC scores Figure 4. Begg’s funnel plots for assessment of publication bias about AIT for ABC scores Figure 4. Begg’s funnel plots for assessment of publication bias about AIT for ABC scores Figure 5. Begg’s funnel plots for assessment of publication bias about AIT for IQ scores Main result of AIT for the effect of IQ Seven RCTs [21,26–29,31,32] were available to determine the effects of treatment on IQ in AIT group. The pooled result showed that the IQ score was significantly higher in AIT group than that in control group [summary SMD = 0.59, 95%CI = 0.41–0.77, I2 = 0.0%, Pheterogeneity = 0.729]. Figure 3 showed the forest plot of SMD with corresponding 95%CI about IQ scores between AIT group and control group. Six of the 7 RCTs were diagnosed by DSM-IV, and only c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attributi License 4.0 (CC BY). Figure 5. Begg’s funnel plots for assessment of publication bias about AIT for IQ scores Figure 5. Discussion In the present study, we explored the relationship between AIT and the effect of children with ASD using a meta-analysis. Findings from this meta-analysis indicated that children with ASD had significantly lower ABC scores and ATEC scores while received AIT. The result also found that IQ scores in AIT group were significantly higher than that in control group. Through our subgroup analyses, the results were consistent with the overall results. In the previous study [8], negative association was found for adults or children with ASD while receiving AIT using six RCTs. Of their included studies, the results did not report the difference between AIT group and control group in a largest study [34]. A small trial inferred no long-term benefit for ASD when using the treatment of AIT [35]. However, one study obtained an increased in ABC scores in the AIT group at 3 months compared with that in control group [36]. In our meta-analysis, the ABC scores and ATEC scores were significantly lower and continued to decline in AIT group after a period of intervention, while the IQ scores were significantly higher in treatment conditions. Seven of our included studies [21,22,25,27–29,31] indicated that children with ASD had better status in AIT group in the fields of language, social interaction, physical movement, take care of themselves, mood, and sleeping using ABC rating scale. This illustrated that symptoms in children with ASD had improved obviously [37]. g y y As seen in Figure 2, in our whole pooled results, significant between-study heterogeneity was appeared in ABC scores and CARS scores, which is common in meta-analysis [38]. We then performed meta-regression to assess this high heterogeneity with covariates of publication year, case numbers, and different diagnostic criteria. As a result, all the above-mentioned factors were not found to significantly contribute to heterogeneity. However, no individual study involved in our meta-analysis had essential effect on the pooled SMD for ABC scores and ATEC scores when we performed the sensitivity analysis. Furthermore, no publication bias was found in the study. There analyses results suggested that the pooled SMD were stable. gg p Our meta-analysis has the following advantages: first, we performed the first meta-analysis to expound the rela- tion of AIT in children with ASD among Chinese and obtain a positive result. The results were not influenced by geographical area as we only included Chinese children with ASD. Discussion Second, according to our final pooled analysis for each individual study, larger participants of children with ASD were included. And this may strengthen the accurate comparisons between AIT group and control group. Third, no publication bias was found due to Egger’s test and funnel plot, which indicated that our results were stable across included studies. There are several limitations need to be mentioned in the present study. First, although most of the diagnostic criteria of ASD were DSM-IV, different diagnostic criteria were existence in the included RCTs. This may affect the pooled results of SMD and 95%CI. However, sensitivity analysis did not support this opinion. Second, only two RCTs were conducted to assess the association for CARS scores and ATEC scores using the treatment of AIT, and we found a negative relationship in CARS scores. Hence, more related RCTs are wanted to confirm AIT in children with ASD in CARS and ATEC rating scale. Third, two RCTs with no training, 1 with music therapy, 6 with routine rehabilita- tion training, 2 with guided education training and 2 with comprehensive treatment were used in the control group. Different treatment of the control group could increase the heterogeneity between studies. In our meta-analysis, we found significant heterogeneity in the results of ALT for ABC scores. Therefore, different treatment of the control group may be an influencing factor on the significant heterogeneity. In summary, AIT can reduce the score of ABC and ATEC and can increase the IQ score among children with ASD in Chinese. Therefore, it is recommended for Chinese children with ASD to receive AIT. 1 article using classification of Chinese mental disorders and diagnostic criteria-3th (CCMD-3) diagnostic criteria. A positive association [summary SMD = 0.59, 95%CI = 0.39–0.79, I2 = 0.0%, Pheterogeneity = 0.608] was found in the subgroup analysis of CCMD-3 diagnostic criteria. 1 article using classification of Chinese mental disorders and diagnostic criteria-3th (CCMD-3) diagnostic criteria. A positive association [summary SMD = 0.59, 95%CI = 0.39–0.79, I2 = 0.0%, Pheterogeneity = 0.608] was found in the subgroup analysis of CCMD-3 diagnostic criteria. g p y g To investigate the influence of each individual study involved in the current analysis on the pooled SMD for IQ scores, we removed studies in sequence. The results were not materially altered, suggesting that the pooled SMD were stable and robust. Publication bias was assessed by Begg’s funnel plot and Egger’s linear regression test. In our meta-analysis, Begg’s test (Pr > \|z\| = 0.368) and Egger’s test (P > \|t\| = 0.835) show no obvious evidence of publication bias. Figure 5 showed the funnel plot also supported the conclusion too. Main result of AIT for the effect of CARS Begg’s funnel plots for assessment of publication bias about AIT for IQ scores Main result of AIT for the effect of IQ Main result of AIT for the effect of IQ Seven RCTs [21,26–29,31,32] were available to determine the effects of treatment on IQ in AIT group. The pooled result showed that the IQ score was significantly higher in AIT group than that in control group [summary SMD = 0.59, 95%CI = 0.41–0.77, I2 = 0.0%, Pheterogeneity = 0.729]. Figure 3 showed the forest plot of SMD with corresponding 95%CI about IQ scores between AIT group and control group. Six of the 7 RCTs were diagnosed by DSM-IV, and only Main result of AIT for the effect of IQ Seven RCTs [21,26–29,31,32] were available to determine the effects of treatment on IQ in AIT group. The pooled result showed that the IQ score was significantly higher in AIT group than that in control group [summary SMD = 0.59, 95%CI = 0.41–0.77, I2 = 0.0%, Pheterogeneity = 0.729]. Figure 3 showed the forest plot of SMD with corresponding 95%CI about IQ scores between AIT group and control group. Six of the 7 RCTs were diagnosed by DSM-IV, and only 7 c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 Funding The authors declare that there are no sources of funding to be acknowledged. Author Contribution N.L. conceived and designed the study. N.L. and L.L. participated in data collecting. Z.T.G. analyzed data. N.L. commented on drafts of the paper. N.L., L.L., Z.T.G, and G.M.L. approved the final manuscript. Competing Interests p g The authors declare that there are no competing interests associated with the manuscript. 8 c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) c⃝2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2018) 38 BSR20181412 https://doi.org/10.1042/BSR20181412 References (2011) Auditory integration training and other sound therapies for autism spectrum disorders (ASD). Cochrane Database Syst. Rev. CD003681 erati, A., Tetzlaff, J., Altman, D.G. and Group, P. 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https://openalex.org/W2888924693	http://eprints.nirt.res.in/1643/1/201867.pdf	English	null	Induction of circulating T follicular helper cells and regulatory T cells correlating with HIV-1 gp120 variable loop antibodies by a subtype C prophylactic vaccine tested in a Phase I trial in India	PloS one	2,018	cc-by	8,969	RESEARCH ARTICLE OPEN ACCESS ‡ These authors also contributed equally to this work. * h @ i t i ‡ These authors also contributed equally to this work. Citation: Munusamy Ponnan S, Swaminathan S, Tiruvengadam K, K. K. V, Cheedarla N, Nesakumar M, et al. (2018) Induction of circulating T follicular helper cells and regulatory T cells correlating with HIV-1 gp120 variable loop antibodies by a subtype C prophylactic vaccine tested in a Phase I trial in India. PLoS ONE 13(8): e0203037. https://doi.org/ 10.1371/journal.pone.0203037 Abstract A Phase I HIV-1 vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009 to test a subtype C prophylactic vaccine in a prime-boost regimen comprising of a DNA prime (ADVAX) and MVA (TBC-M4) boost. The trial demon- strated that the regimen was safe and well tolerated and resulted in enhancement of HIV- specific immune responses. Preliminary observations on vaccine-induced immune responses were limited to analysis of neutralizing antibodies and IFN-γ ELISPOT response. The present study involves a more detailed analysis of the nature of the vaccine-induced humoral immune response using specimens that were archived from the volunteers at the time of the trial. Interestingly, we found vaccine induced production of V1/V2 and V3 region- specific antibodies in a significant proportion of vaccinees. Variable region antibody levels correlated directly with the frequency of circulating T follicular helper cells (Tfh) and regula- tory T cells (Treg). Our findings provide encouraging evidence to demonstrate the immuno- genicity of the tested vaccine. Better insights into vaccine-induced immune responses can aid in informing future design of a successfulHIV-1 vaccine. Editor: David Joseph Diemert, George Washington University School of Medicine and Health Sciences, UNITED STATES Received: March 20, 2018 Accepted: August 7, 2018 Published: August 29, 2018 Copyright: © 2018 Munusamy Ponnan 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: The vaccine trial and the present study were supported and coordinated by the International AIDS Vaccine Initiative (IAVI). IAVI’s work is made possible by generous support from many donors including the Bill & Melinda Gates Induction of circulating T follicular helper cells and regulatory T cells correlating with HIV-1 gp120 variable loop antibodies by a subtype C prophylactic vaccine tested in a Phase I trial in India Sivasankaran Munusamy Ponnan1☯¤, Soumya Swaminathan1☯, Kannan Tiruvengadam1‡, Vidyavijayan K. K.1, Narayana Cheedarla1, Manohar Nesakumar1‡, Sujitha Kathirvel1‡, Rajat Goyal2‡, Nikhil Singla2‡, Joyeeta Mukherjee2‡, Philip Bergin3‡, Jakub T. Kopycinski3‡, Jill Gilmour3, Srikanth Prasad Tripathy1‡, Hanna Elizabeth Luke1☯¤* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 National Institute for Research in Tuberculosis (Indian Council of Medical Research), Chennai, India, 2 International AIDS Vaccine Initiative, New Delhi, India, 3 IAVI Human Immunology Laboratory, Imperial College, London, United Kingdom ☯These authors contributed equally to this work. ¤ Current address: Department of HIV/AIDS, National Institute for Research in Tuberculosis, Chetpet, Chennai, India. Immunogenicity of a HIV-1 subtype C vaccine 68,000 deaths due to AIDS-related illnesses [1]. The increasing burden of HIV presents the urgent need for a vaccine to curb the pandemic. Although several vaccine candidates have been tested in various clinical trials, we are still not close to a successful HIV vaccine [2]. The RV144 trial conducted by the Thai government and the US Military has been the most promis- ing thus far [3].This trial employed a prime-boost vaccination regimen comprising of a non- replicating recombinant canary pox vector ALVAC-HIV (vCP1521) prime and AIDSVAX gp120 B/E boost, and demonstrated that induction of antibodies to the V1/V2 peptides of the HIV-1 envelope correlated with a lower risk of infection, thus becoming the first large-scale Phase III HIV vaccine trial to exhibit a modest level of protective efficacy [4,5]. Foundation, the Ministry of Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan in partnership with The World Bank, the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency for Development Cooperation (NORAD), the United Kingdom Department for International Development (DFID), and the USAID. The full list of IAVI donors is available at www.iavi. org. This study was made possible by the generous support of the American people through funding from United States Agency for International Development (USAID; Grant ID: 2233). The content is the responsibility of the authors and does not necessarily reflect the views of USAID or the United States Government. In 2009, the National Institute for Research in Tuberculosis (formerly Tuberculosis Research Centre) at Chennai, India, and the National AIDS Research Institute at Pune, India, undertook an IAVI-sponsored Phase I HIV-1 subtype C prophylactic vaccine trial, known as the P001 trial (Clinical Trial registry CTRI/2009/091/000051) [6]. This randomized, placebo controlled, dou- ble blind, phase I trial enrolled 16 HIV-uninfected, healthy male and female adult participants at each of the 2 sites. The trial tested the safety and immunogenicity of a heterologous prime- boost regimen employing ADVAX, a DNA-based vaccine consisting of Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes cloned into the pVAX1 mammalian expression vector (Lot # 04030248, Vical, Inc., San Diego, CA) as the prime, and TBC-M4 a recombinant (MVA) vector encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes (Lot # 1B, Therion Biologics Corporation, Cambridge MA) as the boost, with that of homologous MVA alone. Preliminary investigations found that 3 months after the final booster dose, all volunteers in both the groups had positive HIV-specific antibody responses against the Env, Gag, and Pol proteins. The study also characterized the neutralization ability of the antibodies and demon- strated the presence of neutralizing antibodies capable of neutralizing Tier-1 but not Tier-s pseudo viruses at 14 days post final vaccination. However, the titers decreased at 3 months post last vaccination. Based on these observations it was concluded that the vaccine did not induce production of potent broadly neutralizing antibodies [6]. Competing interests: None of the authors of this paper has any conflict of interest. Most HIV-infected subjects produce neutralizing antibodies (NAbs) in low titers with lim- ited neutralizing activity [7,8].Though NAbs tend to protect against infection, constant muta- tions in the virus helps them to evade neutralization by these antibodies [9]. A very small proportion of HIV-infected individuals produce Nabs that can cross-neutralize a large number of viral strains. These antibodies, called bNAbs, have been isolated from individuals referred to as elite neutralizers [10, 11].Non-neutralizing antibodies (n-NAbs) also possess antiviral prop- erty, but their role in preventing infection is still naïve. HIV-1 specific Fc-gamma receptors of vaccine-induced antibodies activate mediators of antibody dependent cellular cytotoxicity (ADCC). Studies have shown that elite responders develop stronger and broader ADCC or neutralizing responses that are also potent against HIV-1 mutants. Recent studies have reported that elite controller-derived cells interact strongly with B cells promoting their maturation, and class switch recombination [12].Tfh cells are a subset of CD4 +T cells defined by CXCR5hi PD-1hi expression that reside in the lymphoid follicles [13, 14] and provide help to B cells for affinity maturation and differential response to pathogens [15]. Interaction of Tfh cells with high-affinity germinal center B cells is crucial for B cell survival, affinity maturation, and antibody class switching. Interleukin-21 (IL-21) secreted by Tfh cells is required for the generation of memory B cells and plasma cells (PCs). IL-21 synergistically acts with CD40L to activate B cells [16, 17]. High frequency of functional circulating PD- 1+CXCR3−CXCR5+ memory peripheral Tfh (pTfh) cells have been reported to correlate with high titres of HIV-specific broadly neutralizing antibodies (bNAbs) and reduced viral loads in HIV-infected individuals [18]. Introduction According to the recent UNAIDS report, there are 36.7 million people living with HIV world- wide. India alone has 2.1 million people living with HIV and has reported approximately 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Ethics statement The P001 vaccine trial was conducted with the approval of the Institutional Ethics Committee of NIRT and samples were collected and stored appropriately with the written informed con- sent of the volunteers. Samples archived during the conduct of the trial for future studies were used for the present study with the approval of the NIRT IEC [NIRT IEC N0-2015013]. Interestingly, the RV144 vaccine, which induced production of V1/V2 antibodies that correlated with protection against HIV infection, also induced IL-21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 2 / 16 Immunogenicity of a HIV-1 subtype C vaccine secreting pTfh cells in the vaccinees [19]. Thus, measuring vaccine-induced pTfh and B cell responses may provide key insights into the ability of the vaccine to elicit antibody-mediated protective immune responses. Regulatory T (Treg) cells down regulate the hyperactive immune system and act as immuno suppressors during HIV infection. Besides, they also regulate peripheral tolerance. Tregs can interact with microbial pathogens to sustain the delicate balance between the host and the pathogen [20–22].Studies have documented that increased frequency of Tregs dampen HIV- specific immune responses [23].The naïve and effector Treg subsets possess unique functions in auto-immunity, immune surveillance, and disease progression [24–25]. However, the func- tional heterogeneity of Treg subsets and their involvement in immune-regulation during HIV vaccination remains largely unexplored and can provide valuable insights to improve vaccine immunogenicity. The present study describes a more detailed characterization of the humoral immune responses induced by the P001 vaccine. PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Study samples Plasma and cryopreserved peripheral blood mononuclear cells (PBMCs) archived at NIRT alone were accessible and used for the present study. The samples belonged to 16 volunteers (9 males and 7 females) who were randomly assigned to either group A or B, with eight partici- pants in each group. Group A participants received two intramuscular (I.M.) injections of ADVAX (a DNA vaccine), or placebo at baseline (time ‘0’) and 1 month, followed by two I.M. injections of TBC-M4 (a recombinant MVA), or placebo at months 3 and 6. Group B partici- pants received three I.M. injections of TBC-M4 or placebo at time 0, month 1 and month 6. Placebo used was 1X sterile PBS with 10% glycerol. Among the 8 volunteers in each group, 6 received the vaccine and 2 received placebo. Fig 1 depicts the schema of the vaccination strategy. Fig 1. Vaccination schedule of IAVI Phase-I prime boost HIV-1 Subtype-C prophylactic vaccine Trial- NIRT-ICMR (P001 Trial). https://doi.org/10.1371/journal.pone.0203037.g001 Fig 1. Vaccination schedule of IAVI Phase-I prime boost HIV-1 Subtype-C prophylactic vaccine Trial- NIRT-ICMR (P001 Trial). https://doi.org/10.1371/journal.pone.0203037.g001 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Immunogenicity of a HIV-1 subtype C vaccine Evaluation of vaccine-induced T and B cells subsets by multicolour immunophenotyping Cryopreserved PBMC that were collected one week after second vaccination and after 1, 2 and 48 weeks after last vaccination were used for this analysis. Cells were thawed, washed with 10% CRPMI and viable cell count was determined using the trypan blue dye exclusion method. cell viability was >90% and recovery was >70%. The cells cells were rested overnight, stained with an amine-reactive viability dye (Live/Dead aqua, Life Technologies) for 20 minutes at room tempera- ture (Chowdhury et al 2015) [26], washed with FACS buffer and stained with the following cock- tail of monoclonal antibodies: T follicular helper cell (Tfh) panel: CD3-APC H7, CD4-BUV737, CD45RO-BUV395, CCR7-PEcy7, CXCR3-APC Alexa 700, CXCR5-BB515, and PD-1-PE; B cell panel: CD3-APC H7, CD38-APC, CD20-PE, CD19-BUV395, IgD-BUV737, CD27- BB515; regu- latory T cell (Treg) panel: CD3-APC H7, CD4-BUV737, CD45RO-BUV395, CCR7-PEcy7, CD25-APC and CD127-PECF 594, for 20 minutes at 4 ºC (antibody and clone description are provided in S1 Table). About 2 × 106 cells were stained for each panel. After staining, the cells were washed, fixed with BD Cytofix (2% paraformaldehyde) and analyzed on a FACS ARIA III flow cytometer (Becton Dickinson). A minimum of 1,000,000 total events were acquired and data were analyzed using FlowJo software, version 10 (Tree star Inc., Ashland, Oregon, USA). Analysis of anti-V1, V2, V3 and MPER-specific antibodies Plasma samples collected at baseline as well as at 2 weeks after the first and last vaccination time point were tested for the presence of IgG antibodies specific to V1, V2, V3 and MPER peptides of HIV-1 envelope gp120 using direct ELISA. Briefly, 96 well plates were coated with 5 μg/ml of peptide pools (15-mer peptides with 11 amino acid overlap) constituting the V1, V2, V3 and MPER regions, in 100 mM NaHCO3 (pH 9.6), by overnight incubation at 4˚C. Plates were washed with PBST (PBS containing 0.05% Tween 20) and blocked with PBS (Lonza, India) containing 1% BSA and 0.05% Tween 20 at 37˚C for 2 hours. After washing, heat-inactivated plasma samples diluted 1:50 with diluent (ABL Inc., MD) were added to each well and incubated at 37˚C for 1 hour. Subsequently, plates were washed and incubated with HRP-conjugated goat anti-human IgG (Thermo Fisher, Waltham, MA) at a dilution of 1:120000 at 37˚C for 1 hour. Plates were developed using One-step TMB substrate (Thermo Fisher). The reaction was stopped using 1N H2SO4 and plates were read at 450nm using a microplate reader (ELx808-BioTek). All tests were performed in duplicates. Pooled normal healthy plasma was used as the negative control. The cut-off for a positive response was defined as mean OD of the negative samples plus 3 times the standard deviation (Table 1). Induction of HIV-1 gp120 variable loop specific antibody responses Plasma samples obtained prior to vaccination as well as at 2 weeks post first and last MVA vac- cination were analyzed for IgG antibodies specific to the first three variable loops (V1, V2 and V3) of the HIV-1 envelope as well as the MPER region. While none of the samples had binding antibodies at baseline or immediately after first vaccination, variable loop antibodies were detected after MVA vaccination (Fig 2). The antibody levels increased consistently with each booster dose of MVA and reached peak values immediately after the last booster (S2 Table). Antibodies to the V1 peptide were detected among 40% (2/6) of Group A and 50% (3/6) of Group B volunteers. Antibodies to V2 peptide were detected among 50% (3/6) of Group A and 40% (2/6) of Group B volunteers. V3-specific antibodies were detected in 90% (5/6) of Group A and Group B volunteers. On the other hand, MPER-binding antibodies were detected only in 10% (1/6) of Group A and Group B volunteers. These findings reveal that the poxvirus and adenovirus based HIV vaccines were capable of inducing antibodies with variable regions binding specificity similar to that seen in the previous HIV-1 vaccine trials [27, 28]. Statistical analysis Statistical analyses were performed using GraphPad Prism, version 5 (GraphPad Software, Inc., CA). Values are presented as median, interquartile, and percentage. Two-way ANOVA Table 1. Variable region and MPER peptides used to study the reactivity of plasma specimens from the vaccinees and placebo recipients. (15-mer peptides with 11 amino acid overlap). V1 CRNVSLCVTLECRNVSSNGTLECRNVSSNGTYNETLECRNVSSNGTYNETRNVSSNGTYNETYNEISNGTYNETYNEIKNCSYNETYNEIKNC V2 SFNATYNEIKNCSFNATTVLRKNCSFNATTVLRDRKQFNATTVLRDRKQTVYAVLRDRKQTVYALFYRRKQTVYALFYRLDIVVYALFYRLDIVPLNK FYRLDIVPLNKKNSSDIVPLNKKNSSENSSLNKKNSSENSSEYYRKNSSENSSEYYRLINC V3 CTRPNNNTRIVCTRPNNNTRKSIRRPNNNTRKSIRIGPGTRKSIRIGPGQTFYSIRIGPGQTFYATGDGPGQTFYATGDIIGDTFYATGDIIGDIRQAT GDIIGDIRQAHC MPER LDERNEKDLLALDNWKNKDLLALDNWKNLWSWALDNWKNLWSWFDITWKNLWSWFDITNWLWWSWFDITNWLWYIRIDITNWLWYIR https://doi.org/10.1371/journal.pone.0203037.t001 Statistical analyses were performed using GraphPad Prism, version 5 (GraphPad Software, Inc., CA). Values are presented as median, interquartile, and percentage. Two-way ANOVA Table 1. Variable region and MPER peptides used to study the reactivity of plasma specimens from the vaccinees and placebo recipients. (15-mer peptides with 11 amino acid overlap). V1 CRNVSLCVTLECRNVSSNGTLECRNVSSNGTYNETLECRNVSSNGTYNETRNVSSNGTYNETYNEISNGTYNETYNEIKNCSYNETYNEIKNC V2 SFNATYNEIKNCSFNATTVLRKNCSFNATTVLRDRKQFNATTVLRDRKQTVYAVLRDRKQTVYALFYRRKQTVYALFYRLDIVVYALFYRLDIVPLNK FYRLDIVPLNKKNSSDIVPLNKKNSSENSSLNKKNSSENSSEYYRKNSSENSSEYYRLINC V3 CTRPNNNTRIVCTRPNNNTRKSIRRPNNNTRKSIRIGPGTRKSIRIGPGQTFYSIRIGPGQTFYATGDGPGQTFYATGDIIGDTFYATGDIIGDIRQAT GDIIGDIRQAHC MPER LDERNEKDLLALDNWKNKDLLALDNWKNLWSWALDNWKNLWSWFDITWKNLWSWFDITNWLWWSWFDITNWLWYIRIDITNWLWYIR https://doi.org/10.1371/journal.pone.0203037.t001 Table 1. Variable region and MPER peptides used to study the reactivity of plasma specimens from the vaccinees and placebo recipients. (15-mer peptides with 11 amino acid overlap). V1 CRNVSLCVTLECRNVSSNGTLECRNVSSNGTYNETLECRNVSSNGTYNETRNVSSNGTYNETYNEISNGTYNETYNEIKNCSYNETYNEIKNC V2 SFNATYNEIKNCSFNATTVLRKNCSFNATTVLRDRKQFNATTVLRDRKQTVYAVLRDRKQTVYALFYRRKQTVYALFYRLDIVVYALFYRLDIVPLNK FYRLDIVPLNKKNSSDIVPLNKKNSSENSSLNKKNSSENSSEYYRKNSSENSSEYYRLINC V3 CTRPNNNTRIVCTRPNNNTRKSIRRPNNNTRKSIRIGPGTRKSIRIGPGQTFYSIRIGPGQTFYATGDGPGQTFYATGDIIGDTFYATGDIIGDIRQAT GDIIGDIRQAHC MPER LDERNEKDLLALDNWKNKDLLALDNWKNLWSWALDNWKNLWSWFDITWKNLWSWFDITNWLWWSWFDITNWLWYIRIDITNWLWYIR https://doi.org/10.1371/journal.pone.0203037.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 4 / 16 Immunogenicity of a HIV-1 subtype C vaccine was used to examine the difference in frequency (%) of different immune cell subsets during the vaccination period. Bonferroni post hoc test was used for sub-group analysis. Correlation analysis was performed to determine the relationship between frequency of different immune cell types and variable region specific antibody responses (OD values). For all analyses, differ- ences were considered significant if p value was <0.05. PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Vaccine-induced plasma B cells Plasma cells were identified as CD19+ CD38+ CD27+ cells, memory cells were distinguished based on expression of IgD and CD27 as described by Kaminski et al. [29], class switched memory cells were identified as IgD- CD27+ cells, IgM memory cells as IgD+ CD27+ cells, double negative memory B cells as IgD- CD27- cells, and mature B cells as IgD+ CD27- cells (S2 Fig). At the pre-vaccination time point, all volunteers had comparable numbers of circulat- ing plasma B cells (Fig 3B). Vaccination induced a significant increase in the number of Plasma B cells particularly in group B (p<0.01). The median frequency of Plasma cells was 1.7% (range: 1.11–2.14%) in Group A, 2.0% (range: 1.32–3.01%) in Group B and 0.5% (range: 0.49–0.79%) in the placebo. At the final time point analyzed, that is 48 weeks post vaccination, the increase appeared to be no longer significant (S3 Table). In addition, a higher frequency of memory B cells, including class-switched memory cells, double negative cells and IgM mem- ory cells was found in Group B as compared to Group A, although the increase was not statisti- cally significant (S3 Fig). Induction of circulating T follicular helper cells correlating with levels of variable loop antibodies PBMC from the pre-vaccination, one week post second vaccination and 1, 2 and 48 weeks post final vaccination time points were analyzed by multicolor flow cytometry to enumerate the Fig 2. Reactivity of plasma obtained volunteers at two weeks after last vaccination to the variable loop and MPER peptides. (Note: 2wk+Post VAC-3 –two weeks after the last MVA booster dose).Values represent ELISA-generated optical density (OD) values at wavelength 405nM. Plasma was tested at a dilution of 1:50. Two-way ANOVA using Bonferroni post-hoc test was used at 5% level of significance. https://doi.org/10.1371/journal.pone.0203037.g002 Fig 2. Reactivity of plasma obtained volunteers at two weeks after last vaccination to the variable loop and MPER peptides. (Note: 2wk+Post VAC-3 –two weeks after the last MVA booster dose).Values represent ELISA-generated optical density (OD) values at wavelength 405nM. Plasma was tested at a dilution of 1:50. Two-way ANOVA using Bonferroni post-hoc test was used at 5% level of significance. Fig 2. Reactivity of plasma obtained volunteers at two weeks after last vaccination to the variable loop and MPER peptides. (Note: 2wk+Post VAC-3 –two weeks after the last MVA booster dose).Values represent ELISA-generated optical density (OD) values at wavelength 405nM. Plasma was tested at a dilution of 1:50. Two-way ANOVA using Bonferroni post-hoc test was used at 5% level of significance. https://doi.org/10.1371/journal.pone.0203037.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 5 / 16 Immunogenicity of a HIV-1 subtype C vaccine number of pTfh cells (S1 Table) defined as CD4+CD45RO+CCR7+PD1+CXCR5+CXCR3- cells as described by Locci et al [18] (S1 Fig). Vaccination induced significantly more Tfh cells than did the placebo (Fig 3A). The median frequency of Tfh cells among CD4+ T cells was 0.13% (range: 0.061–0.17%) in Group A, 0.2% (range: 0.72–0.2%) in Group B and 0.08% (range: 0.04–0.15%) in the placebo (S3 Table). The numbers of Tfh cells were observed to increase progressively with time in both groups A and B, and remained significantly higher in the vaccine recipients as compared to the placebo (p<0.01). The frequency of circulating memory-like Tfh cells in Group B was significantly higher than in Group A at all the time points (p<0.01; S3 Table). Interestingly, a positive cor- relation was observed between the V2 and V3 specific antibody response and the number of circulating Tfh cells (p<0.033 and p<0.017 respectively) in Group A alone (Fig 4). PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Induction of regulatory T cells correlating with levels of variable loop antibodies Tregs were defined as CD3+ CD4+ CD25+ CD127- cells; memory cells were identified based on the expression of CCR7 and CD45RO as described by Baecher et al.[30] and Su et al. [31]; activated Tregs were defined as CD3+ CD4+ CD25+ CCR7- CD45RO+ cells, and resting Tregs cells were defined as CD3+ CD4+ CD25+ CCR7+ CD45RO+ cells (S4 Fig). While there was no difference in the frequency of Tregs between placebo and vaccinees prior to vaccina- tion (Fig 3C), vaccination resulted in a significant induction of Tregs. Significantly higher numbers of Tregs (p<0.001) were seen at all time points post-vaccination). The median fre- quency of Tregs among CD4+ T cells was 1.2% (range 0.84–1.59%) in Group A, 2.0% (range: 0.5–2.25%) in Group B and 0.5% (range: 0.04–0.71%) in the placebo (S3 Table). Group B vol- unteers had a higher frequency of Tregs as compared to Group A at 1, 2 and 48 weeks post final MVA vaccination (p<0.012, p<0.009 and p<0.003 at the three time points respectively; S3 Table). We further analyzed the proportion of activated and resting Tregs among the vacci- nated individuals and found that the proportion of activated Tregs was high at the time of last vaccination, but declined subsequently with time. The median frequency of activated Tregs among CD4+ T cells was 0.49% (range: 0.22–0.19%) in Group A, 1.125% (range 0.66–2.0%) in Group B and 0.8% (range: 0.25–1.19%) in the placebo. In contrast, the frequency of resting Tregs increased significantly within a week after final vaccination (Data not shown). We observed a positive correlation between the number of Treg cells and V1 and V3 specific PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 6 / 16 Immunogenicity of a HIV-1 subtype C vaccine PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 7 / 16 Immunogenicity of a HIV-1 subtype C vaccine Fig 3. Vaccine induced immune cell subsets in PBMC samples (Note: 2V - First MVA, 2V+1wk—one week after first MVA vaccination, 3V - Last MVA booster dose, 3V+1wk—one week after final MVA booster dose, 3V+2wk— two weeks after final MVA dose, 3V+48wk—48 weeks after final MVA booster). (3A) Frequency of vaccine induced circulating T follicular helper cells in Groups A and B after 2nd vaccination, 1-week post 2nd vaccination, 3rd vaccination, and 1st week, 2nd week, and 48th week post 3rd vaccination. Induction of regulatory T cells correlating with levels of variable loop antibodies (3B) Frequency of Plasma B cells in Groups A and B after 2nd vaccination, 1st week post 2nd vaccination, 3rd vaccination, 1st week, 2nd week and 48 weeks post 3rd vaccination (3C) Frequency of Circulating Regulatory cells T cells in Groups A and B after 2nd vaccination, 1st week post 2nd vaccination, 3rd vaccination, 1st week, 2nd week and 48 weeks post 3rd vaccination. Graphical representation of the frequency of immune cells in placebo and vaccinees of both groups at different time points. Horizontal bars represent mean values and P values were calculated using two-way ANOVA using Bonferroni post hoc test. https://doi.org/10.1371/journal.pone.0203037.g003 antibody levels in the vaccinated individuals (p<0.035 in group A and p<0.002 in Group B respectively) (Fig 4). antibody levels in the vaccinated individuals (p<0.035 in group A and p<0.002 in Group B respectively) (Fig 4). PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Discussion Preliminary analy- sis revealed that vaccination resulted in production of HIV-specific antibody and IFN-γ responses, and reported that though the initial response appeared to be significantly higher in the DNA prime/MVA boost group (Group A) as compared to the MVA alone group (group B), the effect lasted only for a short time, implying that both the heterologous DNA/MVA prime-boost and the homologous MVA regimens were immunologically comparable [6]. Here we describe our findings on vaccine-induced humoral immune responses studied using PBMC and plasma samples of the trial participants that were archived under ideal condi- tions at NIRT. The original study reported the induction of neutralizing antibodies with very limited cross-neutralization activity. Prompted by the findings of the RV144 trial, we deter- mined the ability of the vaccine to induce V1/V2 specific antibodies, as well as antibodies to the V3 and MPER peptides of HIV-1. While none of the samples had HIV-specific antibodies binding to the variable regions or MPER peptides at baseline or immediately after first vacci- nation, we observed V1/V2 and V3-specific antibodies in a significant proportion of the volun- teers in both the groups. On the other hand, only a small proportion of the volunteers developed MPER-specific antibody responses. Our findings are in line with previous observa- tions that poxvirus and adenovirus-based HIV vaccines are capable of inducing antibodies to the variable regions of HIV-1 envelope. An earlier study in macaques reported a 10-fold increase in the level of anti-env antibodies following immunization with MVA alone as com- pared to those who were immunized with DNA and MVA [40]. A similar pattern was also observed in the trial under discussion, when total anti-env antigen specific antibody responses were measured [6]. A closely related trial called the P002 trial conducted in UK with the same vaccine construct used in the present trial, also reported higher titres of HIV-specific antibod- ies targeting rgp41 Env, rgp140 Env, p24 Gag, and rgp120 Env proteins in Group B than in Group A volunteers [41]. The variable loops of the HIV-1 envelope are known to be highly immunogenic in both HIV and SIV [42–43], and possess contact points for the binding of bNAbs and non-neutralis- ing antibodies (nnAbs) [18, 19]. Multiple monoclonal bNABs targeting the V1/V2 domain (e.g. PG9, PG16, CH01, CH03, and PGT145) have been isolated from HIV-infected individuals [19, 44, 45]. Discussion The search for an effective HIV vaccine is still on. Some of the popular HIV vaccine trials con- ducted in the past including the VAX004, Merck 023 and HTN505 trials, have shown that it is difficult to elicit high titers of HIV-1-Nabs, and that the low levels of antibodies induced by the vaccines do not last long in vaccinated individuals [32]. The RV144 vaccine trial has thus far been the only HIV vaccine trial that brought some hope that a preventive HIV vaccine is possi- ble. The trial demonstrated 31.2% reduction in risk of HIV infection among individuals vacci- nated with live recombinant ALVAC-HIV(R) (vCP1521) and VaxGen gp120 B/E (AIDSVAX (R) B/E) in a prime-boost regimen [32]. A large number of immunological studies were under- taken to understand the correlates of vaccine-induced protection against HIV infection in the trial participants [33–36]. One of the interesting observations of these studies was that IgG antibodies that bind to the V1/V2 region of the HIV-1 Env correlated with lower infection rates among vaccinees [37–39]. The present study aimed to characterize the immunogenicity of the subtype C prophylactic vaccine that was tested by the Indian Council of Medical Research (ICMR) and the International AIDS Vaccine Initiative (IAVI) using MVA Fig 4. Correlation between immune cells and variable loop and MPER-specific antibodies. (A) Frequency of vaccine induced circulating T follicular helper cells vs antibodies to the V1 antigen. (B) Frequency of vaccine induced circulating T follicular helper cells vs antibodies to the V3 antigen. (C) Frequency of vaccine induced circulating T regulatory cells vs antibodies to the V2 antigen. (D) Frequency of vaccine induced circulating T regulatory cells vs antibodies to the V3 antigen. https://doi org/10 1371/journal pone 0203037 g004 Fig 4. Correlation between immune cells and variable loop and MPER-specific antibodies. (A) Frequency of vaccine induced circulating T follicular helper cells vs antibodies to the V1 antigen. (B) Frequency of vaccine induced circulating T follicular helper cells vs antibodies to the V3 antigen. (C) Frequency of vaccine induced circulating T regulatory cells vs antibodies to the V2 antigen. (D) Frequency of vaccine induced circulating T regulatory cells vs antibodies to the V3 antigen. https://doi.org/10.1371/journal.pone.0203037.g004 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Immunogenicity of a HIV-1 subtype C vaccine (TBC-M4) and ADVAX as vaccine candidates in India a few years back [6]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Discussion However, induction of bNAbs through vaccination has remained a major chal- lenge. While earlier studies reported that production of HIV specific bNAb correlated with clinical and viral parameters [46], most individuals with high viral loads do not develop bNAb, making it evident that the synergistic action of various factors is required for the development of bNAbs [47]. The antibodies generated in the P001 trial were found to possess very limited neutralizing activity. However, their ability to bind to gp120 peptides suggests a possible role in stimulating other protective mechanisms such as ADCC, and/or cell mediated virus inhibi- tion [48]. Non-neutralizing epitopes of HIV expressed on the surface of infected cells may per- mit the destruction of these cells via ADCC, antibody-dependent cell mediated virus inhibition (ADCVI) or lysis of HIV particles via complement-mediated attack on the virus envelope [49, 50]. The lower rates of infection seen among vaccine recipients who developed HIV-1 specific nnAbs against the V2/V3 peptides in the RV144 trial provide major support for this hypothesis [38] Tfh cells prime the B cells, support affinity maturation and play a pivotal role in antibody production [18, 19, 51–53]. Activation signals from Tfh cells are critical for the development of antibody producing B cells [54]. The frequency of Tfh cells is reported to be higher in the blood and lymphoid organs of those with AIDS as compared to HIV+ non-progressors [55]. The memory subset of Tfh cells that exist in circulation in peripheral blood are referred to as peripheral Tfh (pTfh) cells [18, 19,56–59]. Correlation between the frequency of pTfh cells in circulation and the development of broadly neutralising antibodies against HIV has been PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 9 / 16 Immunogenicity of a HIV-1 subtype C vaccine reported in a large cohort of HIV+ individuals [18]. The present study identified a significantly higher frequency of pTfh cells among vaccine recipients in both Groups A and B, as compared to the placebo. Interestingly, the frequency of pTfh cells correlated positively with levels of var- iable loop antibodies, suggesting that the vaccine primed the host immune system and gener- ated pTfh cells to produce HIV-specific antibodies. Although the vaccine under study failed to elicit a potent bNAb response, it is encouraging to note that the vaccine induced Tfh cells and elicited production of variable loop specific antibodies. Discussion A positive association between Tfh cell frequency, expansion of the B cell compartment and increase in circulating high-avidity SIV- specific antibodies during chronic SIV infection have previously been reported [60], suggest- ing strongly that induction of Tfh cells results in a significant antibody response. However in our study, the correlation was found to be statistically significant only in Group A, although a positive correlation was also observed in Group B. The very small sample size of the present study is a major limitation in this regard. Development of new candidate vaccines for humans against major infectious killers is known to depend largely on the quality of the antibody response. Thus, Tfh cell based vaccines have the advantage of positively influencing the generation of a robust protective immune response in humans [18,55]. Gag-specific pTfh cells are generally thought to provide help in induction of B cells, while Env-specific pTfh help in antibody class-switching [60]. Gag- and Env-specific IL-21+ pTfh cells upregulate the expression of cytolytic markers on HIV-specific CD8+T cells. The RV144 vaccine trial demonstrated that ALVAC+AIDSVAX induced a higher percentage of pTfh cells as compared to ALVAC alone or the DNA-Ad5 regimen like that used in the unsuccessful HVTN 505 trial. Our observations as well as that of others high- light the critical role of pTfh cells in the protective efficacy of a HIV vaccine. higher percentage of pTfh cells as compared to ALVAC alone or the DNA-Ad5 regimen like that used in the unsuccessful HVTN 505 trial. Our observations as well as that of others high- light the critical role of pTfh cells in the protective efficacy of a HIV vaccine. Several studies have shown that the interaction between the germinal center B cells and Tfh cells through numerous surface receptors is essential for the optimal development and survival of both cell types [52, 61]. Long-term immunity to infectious agents, such as viruses, is primar- ily due to the expression of antibodies from plasma cells and antibody secreting cells. Studies in animal models have shown that constant replenishment of plasma cells by memory cells is not mandatory because of the existence of long-lived plasma cells [61]. However, plasma cell longevity and its role in maintaining serum antibody levels are poorly defined. PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Immunogenicity of a HIV-1 subtype C vaccine cells and plasma B cells. We also observed a positive correlation between the number of Treg cells and V1/V3 region antibody response in both the groups. In summary, the present study characterized in detail the nature of the antibody response and detected V1/V2 specific antibody responses in a significant portion of volunteers, which are now known to be positive correlates for protection against HIV infection, through anti- body dependent cell-mediated cytotoxicity (ADCC) or some other yet unidentified mecha- nism [70]. An effort to elucidate the ADCC activity of antibodies elicited by the vaccine would provide more detailed information on the protective nature of antibody response and further guidance for the design of a successful HIV-1 vaccine. Further, the variable loop antibody lev- els correlated positively with induction of circulating pTfh cells and Treg cells. One of the major limitations of this study is the small sample size, comprising of only 16 volunteers including 4 placebo and the remaining 12 distributed between the two study groups. Hence, many of the correlation analyses undertaken here lacked sufficient power to achieve statistical significance although positive responses were seen in many instances. However, the present study has revealed encouraging findings on the potential of the vaccine regimen for inducing favorable immunogenic responses among volunteers. Therefore, these results suggest that an in-depth analysis of vaccine-induced immune response can aid in informing future design of an HIV-1 vaccine based on the Indian Clade C virus. S1 Table. Reagents used for multicolor flow cytometry. (DOCX) S2 Table. Reactivity of plasma to variable region and MPER peptides. Values represent ELISA-generated data optical density (OD) values at a wavelength of 405nM. Plasma was tested at a dilution of 1:50. Statistical analysis was performed using anova. Bonferroni post hoc test was used at 5% level of significance. (DOCX) S3 Table. Comparison of mean frequency of circulating T follicular helper cells, plasma B cells, regulatory T cells in placebo and vaccinees. P values were calculated using Two-way ANOVA using Bonferroni post hoc test.—p<0.05; —p<0.01; —p<0.001. (DOCX) S4 Table. Correlation between immune cells, variable loop, and MPER-specific antibodies. (XLSX) Discussion In the present study, we also measured the frequency of plasma B cells and memory B cells post-vaccination and found that vaccine recipients had significantly higher numbers of plasma B cells than pla- cebo. Group B volunteers had higher frequencies of plasma B cells than Group A vaccinees, suggesting that the homologous regimen was better in terms of plasma B cell and antibody production than the heterologous regimen. Differentiation into plasma cells and germinal cen- ter B cells greatly depends on high-affinity antigens, and Tfh cells are essential for B cell matu- ration, survival, and proliferation [18, 52]. Though the frequency of pTfh cells was found to be higher among the vaccinees, we could not observe any correlation with B cell frequency possi- bly due to the very small sample size. Regulatory T cells (Tregs) are known to suppress activation of multiple cell types including CD4+ and CD8+ T cells, B cells, natural killer cells, and dendritic cells. In HIV infection, stud- ies have reported that Tregs cause significant increase in antigen-specific cytokine production from HIV specific CD4+ and CD8+ T cells [62] and help to prevent reactivation of latent res- ervoirs of HIV [63–66]. Although there is very little data available on the induction of Tregs and their role in vaccination, Tregs are thought to play an important role in down regulating host immune response in order to maintain immune homeostasis and thus impact vaccine efficacy [67–69]. We were curious to see if vaccine under discussion had any effect on this sub- set of cells. Interestingly, we found that both the regimens induced Tregs without compromis- ing the vaccine’s immunogenicity in terms of antibody production as well as induction of pTfh PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 10 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 Acknowledgments We are thankful to Dr. V. D. Ramanathan who was the Principal Investigator of the P001 trial at the National Institute for Research in Tuberculosis, as well as the volunteers and staff who were part of the trial. We wish to particularly thank Mr. Sathyamurthi P. and the research team for making this study possible through carefully preserving the samples collected during the conduct of the Phase I trial. We also acknowledge the scientific and technical inputs pro- vided by Dr. Alena Srinivasan; and Dr. Shweta Chatrath (from International AIDS Vaccine Initiative) for her assistance in technical/language editing, and proofreading of the manuscript. S4 Table. Correlation between immune cells, variable loop, and MPER-specific antibodies. (XLSX) S1 Fig. Representative pseudocolor FACS plot of circulating memory like T Follicular Helper cells. T cells were gated first on lymphocytes and then on memory T cells (CCR7+- CD45RO+) followed by Tfh cells (CXCR5+PD-1+CXCR3-). (DOCX) S2 Fig. Representative Pseudo color FACS plot of B cells and memory subsets. B cells were gated first on lymphocytes and then on plasma cells (CD38+ CD27+) and memory B cells (CD27 and IgD). (DOCX) S3 Fig. Frequency of circulating memory B cell subsets. Graphical representation showing the % of memory B cells in placebo and vaccinees of both groups at different time points. The horizontal bars represent median and dot values represent scatter points. P values were calcu- lated using Two-way ANOVA using Bonferroni post hoc test. /†—p<0.05; /††—p<0.01; PLOS ONE \| https://doi.org/10.1371/journal.pone.0203037 August 29, 2018 11 / 16 Immunogenicity of a HIV-1 subtype C vaccine /†††—p<0.001. (DOCX) /†††—p<0.001. (DOCX) S4 Fig. Representative pseudocolor FACS plot of regulatory T cells. T cells were gated first on lymphocytes and then on Tregs (CD4+CD127dimCD25+) followed by memory Tregs (CCR7+CD45RO+). ( ) References 1. UNAIDS. Prevention gap report. 2016. http://www.unaids.org/sites/default/files/mediaasset/2016- prevention-gap-report_en.pdf. Accessed 6 June 2017. 2. Streeck H, D’souza MP, Littman DR, Crotty S. Harnessing CD4+ T cell responses in HIV vaccine devel- opment. 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Data curation: Sivasankaran Munusamy Ponnan, Jakub T. Kopycinski. Formal analysis: Sivasankaran Munusamy Ponnan, Joyeeta Mukherjee, Jakub T. Kopycinski, Jill Gilmour. Funding acquisition: Soumya Swaminathan, Nikhil Singla, Joyeeta Mukherjee, Philip Bergin, Jill Gilmour, Hanna Elizabeth Luke. Investigation: Sivasankaran Munusamy Ponnan, Soumya Swaminathan, Rajat Goyal, Jakub T. Kopycinski, Hanna Elizabeth Luke. Methodology: Sivasankaran Munusamy Ponnan, Vidyavijayan K. K., Narayana Cheedarla, Sujitha Kathirvel, Philip Bergin, Jakub T. Kopycinski. Project administration: Rajat Goyal, Nikhil Singla, Joyeeta Mukherjee, Jill Gilmour, Srikanth Prasad Tripathy. Resources: Sivasankaran Munusamy Ponnan, Rajat Goyal, Nikhil Singla, Jakub T. Kopycinski. Software: Sivasankaran Munusamy Ponnan, Kannan Tiruvengadam, Philip Bergin, Jakub T. Kopycinski. Supervision: Soumya Swaminathan, Rajat Goyal, Philip Bergin, Jill Gilmour, Srikanth Prasad Tripathy, Hanna Elizabeth Luke. 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https://openalex.org/W2163031497	http://scholarworks.calstate.edu/downloads/736665530	English	null	Search for the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi>b</mml:mi></mml:mrow></mml:msub></mml:math> and other hidden-beauty states in the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si2.gif" overflow="scroll"><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><…	Physics letters. B	2,015	cc-by	24,914	1. Introduction pends on the branching fraction for Xb →π+π−Υ (1S), which is unknown. The π+π−Υ (1S) channel also provides the opportunity to measure the production of the Υ (13D J) states. These have not been observed at the Tevatron; their production cross sections in pp collisions are also unknown, but an early colour-octet cal- culation [17] gives values comparable to that of the Υ (2S). The Υ (13D2) has been observed in radiative transitions by CLEO [18] and BaBar [19]. The X(3872) is the ﬁrst and the best-studied of the new hidden-charm states seen in the last decade. Observed by Belle in decays B± →K ± X(→π+π−J/ψ) [1], it was quickly conﬁrmed by BaBar [2], CDF [3], and DØ [4]. In particular, CDF and DØ found that the X(3872) is produced directly in pp collisions; recently CMS has measured the product of the pp production cross section and the π+π−J/ψ branching fraction to be (6.56 ± 0.29 ± 0.65)% of the value for the ψ(2S) [5]. The mass, narrow width, J PC = 1++ quantum number assignment [6–10], and decay characteristics of the X(3872) make it unlikely to be a conventional quarkonium state, and there is an extensive literature discussing its structure. Weakly bound D0D∗0 molecular models (for example, Refs. [11, 12]) have been popular due to the proximity of the X(3872) to the D0D∗0 threshold; various [qc][¯q¯c] tetraquark (for example, Refs. [13,14]) and other models have also been proposed. The production of Υ (10 860) and some other hidden-beauty states may also be studied using π+π−Υ (1S). The Υ (10 860) de- cay to π+π−Υ (1S) has a surprisingly large partial width [20–22]: current world average results are (0.29 ± 0.15) MeV for the Υ (10 860), to be compared with (0.89 ± 0.08) keV for the Υ (3S), and (1.7 ± 0.2) keV for the Υ (4S) [23]. Belle has also presented evidence of exotic substructure in this decay [24]. The natural widths of the Υ (10 860), Υ (11 020), and other states above the open-beauty threshold are larger than the detector resolution, and must be explicitly considered in any search. Heavy-quark symmetry suggests the existence of a hidden- beauty partner – a so-called Xb state – which should be produced in pp collisions [15]. The molecular model of Swanson [12,16] predicts an Xb mass of 10561 MeV, while tetraquark predictions vary: for example, Ref. a b s t r a c t Article history: Received 16 October 2014 Received in revised form 26 November 2014 Accepted 26 November 2014 Available online 2 December 2014 Editor: W.-D. Schlatter This Letter presents a search for a hidden-beauty counterpart of the X(3872) in the mass ranges of 10.05–10.31 GeV and 10.40–11.00 GeV, in the channel Xb →π+π−Υ (1S)(→μ+μ−), using 16.2 fb−1 of √ s = 8 TeV pp collision data collected by the ATLAS detector at the LHC. No evidence for new narrow states is found, and upper limits are set on the product of the Xb cross section and branching fraction, relative to those of the Υ (2S), at the 95% conﬁdence level using the CLS approach. These limits range from 0.8% to 4.0%, depending on mass. For masses above 10.1 GeV, the expected upper limits from this analysis are the most restrictive to date. Searches for production of the Υ (13D J), Υ (10 860), and Υ (11 020) states also reveal no signiﬁcant signals. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. Search for the Xb and other hidden-beauty states in the π+π−Υ (1S) channel at ATLAS .ATLAS Collaboration ⋆ ⋆E-mail address: atlas.publications@cern.ch. . .055 shed by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by Physics Letters B 740 (2015) 199–217 Physics Letters B 740 (2015) 199–217 ⋆E-mail address: atlas.publications@cern.ch. peaks are used to validate the measurement technique. Results are http://dx.doi.org/10.1016/j.physletb.2014.11.055 0370-2693/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. dx.doi.org/10.1016/j.physletb.2014.11.055 2693/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by 3 1 ATLAS uses a right-handed coordinate system with its origin at the nominal in- teraction point (IP) in the centre of the detector, the x-axis pointing to the centre of the LHC ring, and the z-axis along the beam pipe; the y-axis points upward. Cylindrical coordinates (r, φ) are used in the transverse plane; φ is the azimuthal angle around the beam pipe. Pseudorapidity and transverse momentum are de- ﬁned in terms of the polar angle θ as η = − ln(tanθ/2) and pT = p sinθ. The (η, φ) distance between two particles is deﬁned as R = ( η)2 + ( φ)2. For a particle with momentum ⃗p = (px, p y, pz) and energy E, the rapidity is deﬁned as y = 0.5 ln([E + pz]/[E −pz]). 2. The ATLAS detector In the remainder of the event, dipion candidates are formed from oppositely charged pions with \|ηπ\| < 2.5, each required to have ≥1 pixel hits, ≥6 silicon microstrip hits, and pπ T > 400 MeV; no other requirements (such as lepton vetoes) are imposed. The Υ (1S) candidate and the dipion system are combined by perform- ing a four-track common-vertex ﬁt, with the μ+μ−mass con- strained to m1S, and tracks assigned μ or π masses as appropri- ate. This signiﬁcantly improves the mass resolution: for example, in the Υ (2S) simulation, the RMS improves from 142 MeV to 9.7 MeV. The π+π−Υ (1S) vertex ﬁt is required to have a chi- square less than 20; this is 95% eﬃcient for Υ (2S) decays but reduces background by a factor of ∼10. All remaining π+π−Υ (1S) candidates with invariant masses up to 11.2 GeV are retained. The ATLAS detector [26] is composed of an inner tracking sys- tem, calorimeters, and a muon spectrometer. The inner detector (ID) surrounds the pp interaction point and consists of silicon pixel and microstrip detectors, and a transition radiation tracker, all im- mersed in a 2 T axial magnetic ﬁeld. The ID spans the pseudora- pidity1 range \|η\| < 2.5 and is enclosed by a system of electromag- netic and hadronic calorimeters. Surrounding the calorimeters is the muon spectrometer (MS) consisting of three large air-core su- perconducting magnets (each with eight coils) providing a toroidal ﬁeld, a system of precision tracking chambers, and fast detectors for triggering. Monitored drift tubes and cathode-strip chambers provide precision measurements in the bending plane of muons within the pseudorapidity range \|η\| < 2.7. Resistive plate and thin gap chambers are used to make fast event data-recording decisions in the ranges \|η\| < 1.05 and 1.05 < \|η\| < 2.4 respectively, and also provide position measurements in the non-bending plane and im- prove pattern recognition and track reconstruction. MS momentum measurements are based on track segments formed in at least two of the three precision chamber planes. For a state decaying to π+π−Υ (1S), the acceptance A is de- ﬁned as the fraction of decays where both muons have pμ T > 4 GeV, both pions have pπ T > 400 MeV, and all four particles are within \|η\| < 2.5. 2 An un-prescaled trigger with a pμ T = 6 GeV threshold on the muons was also considered, but found to result in a lower sensitivity using the optimisation proce- dure described in Section 5. 1. Introduction [14] predicts masses of 10 492, 10 593, or 10 682 MeV, depending on the ﬂavour of the light quarks. In 2013, CMS reported [25] the results of their search for Xb → π+π−Υ (1S)(→μ+μ−), ﬁnding no evidence for narrow states in the 10.06–10.31 GeV and 10.40–10.99 GeV mass ranges. They set upper limits on the product of cross section and branching fraction at values between 0.9% and 5.4% of the Υ (2S) rate. The decay Xb →π+π−Υ (1S)(→μ+μ−), analogous to the decay mode in which the X(3872) was discovered, provides a straightforward way to reconstruct an Xb. Any resulting measure- ment or upper limit on the Xb production cross section then de- This Letter presents a search for the Xb and other hidden- beauty states at ATLAS, using a 16.2 fb−1 pp collision data sam- ple collected at √ s = 8 TeV during the 2012 run of the LHC. The analysis is performed simultaneously across eight kinematic bins of varying sensitivity; the Υ (2S) and Υ (3S) →π+π−Υ (1S) peaks are used to validate the measurement technique. Results are ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 200 presented in terms of the product of production cross section and π+π−Υ (1S) branching fraction, relative to that for the Υ (2S). 350 MeV of the Υ (1S) mass [23], m1S, is retained and consid- ered an Υ (1S) →μ+μ−candidate. To conﬁrm that this pair is the same as that used in the trigger, the reconstructed and trigger- level muons are required to match with R < 0.01. 2. The ATLAS detector States with pT < 5 GeV or rapidity \|y\| > 2.4 have very low acceptance, so candidates in these regions (which also suffer from high background) are excluded. 4. Data and simulation samples The ATLAS detector employs a three-level trigger [27] to re- duce the 20 MHz proton bunch collision rate to the few-hundred hertz transfer rate to mass storage. This analysis is based on a Level-1 muon trigger that searches for hit coincidences between muon trigger detector layers inside pre-programmed geometrical windows that bound the path of muon candidates above a given pμ T threshold, and provide a rough estimate of their position, for \|ημ\| < 2.4. There are two subsequent software-based trigger stages, in which muon candidates incorporate, with increasing pre- cision, information from both the MS and the ID, reaching position and momentum resolution close to that provided by the oﬄine re- construction. The techniques adopted in this analysis were developed us- ing √ s = 7 TeV data collected in 2011 and simulation samples generated under the same running conditions, with particular at- tention to Υ (2S) →π+π−Υ (1S) mass and (\|y\|, pT) distributions. Backgrounds due to inclusive Υ (1S) production and combinato- rial μ+μ−sources were studied using μ+μ−sideband and μ±μ± same-sign samples, and found to be featureless above 9.8 GeV. The results presented here are based on data from the 2012 √ s = 8 TeV pp run at the LHC; standard data-quality criteria are used to ensure eﬃcient detector performance. The trigger em- ployed in the event selection was subject to an instantaneous luminosity-dependent prescale factor,2 and provided an integrated luminosity of L = 16.2 fb−1. The resulting data sample includes over 10 million μ+μ−combinations: a ﬁt to the sample ﬁnds (6.00 ± 0.01) × 106 from Υ (1S) decays, (0.200 ± 0.002) × 106 from Υ (2S) decays, and the remainder from combinatorial back- ground. Given the reconstruction and event selection choices de- scribed in the previous section, each dimuon gives rise (on aver- age) to 19.5 π+π−Υ (1S) candidates with invariant mass below 11.2 GeV. A procedure to select at most one candidate per event was considered. However, after the adoption of the binning ap- proach described in Section 5, candidate selection was found to worsen the expected sensitivity to a hypothetical Xb signal. There- fore, all π+π−Υ (1S) candidates are retained for analysis. 3. Reconstruction and event selection Events are selected using a trigger requiring two muons of opposite charge, each with pμ T > 4 GeV, successfully ﬁtted to a common vertex. The μ+μ−mass range accepted by the trigger, 8–12 GeV, includes the Υ (1S), Υ (2S), and Υ (3S) signal peaks. In the oﬄine reconstruction for this analysis, muon reconstruc- tion relies on a statistical combination of an MS track and an ID track. The selected muons are restricted to \|ημ\| < 2.3, ensuring high-quality tracking and a reduction of fake muon candidates. This restriction also removes regions of strongly varying eﬃciency and acceptance. The reconstruction of π+π−Υ (1S) candidates begins with pairs of oppositely charged muon candidates that satisfy the same kine- matic conditions used by the trigger, and have ≥2 pixel and ≥6 silicon microstrip detector hits. Each pair is subjected to a common vertex ﬁt, and a loose chi-square selection is imposed to exclude very poor candidates. Any dimuon with an invariant mass within Simulated samples are used to optimise the selections, model signal decays, develop ﬁtting models, and calculate eﬃciencies. In- dividual samples are used for the Υ (2S), Υ (3S), Υ (13D J) triplet, Υ (10 860), and for two hypothetical Xb masses, 10 233 MeV and 10 561 MeV. Production is modelled with the AU2 [28] tune of Pythia 8.170 [29] and the CTEQ6L1 [30] parton distribution func- tions. In the decay to π+π−Υ (1S), the three-body phase space is uniformly sampled. Isotropic spin alignment of the parent is as- sumed: for Υ (2S) and Υ (3S) this is supported by a recent CMS measurement [31]. Passage of particles through the detector is ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 201 Fig. 1. The π+π−Υ (1S) invariant mass distribution in the kinematic bin most sen- sitive to an Xb signal: \|y\| < 1.2, pT > 20 GeV, and cosθ∗> 0. The only apparent peaks are at the masses of the Υ (2S) (10023 MeV) and Υ (3S) (10355 MeV). simulated with Atlfast II [32], supplementing Geant4 [33,34] with a parameterised calorimeter response. Simulated kinematic distributions of the ﬁnal-state pions and muons are sensitive to mismodelling of the pT and y distributions of the parent state. 5. Fitting strategy Due to the low pion momentum in the π+π−Υ (1S) rest frame, and the reconstruction threshold of pπ T = 400 MeV in the labo- ratory frame, true π+π−Υ (1S) decays are preferentially recon- structed if the parent has large pT or small θ∗(deﬁned as the angle, in the parent rest frame, between the dipion momentum and the lab-frame parent momentum). In background candidates, the dipion and dimuon systems are unrelated, and the dipions typically have low pππ T in the lab; boosting to the π+π−μ+μ− frame yields large values of θ∗, with a broad distribution around cosθ∗= 0. In the (pT, cosθ∗) plane, then, the ratio of signal to background candidates is largest in the upper-right region, and smallest in the lower-left. y pT cos θ The shape of the signal peaks reﬂects the detector resolution, which differs between the barrel and endcap, and varies as a func- tion of mass; in a given rapidity bin at a given mass, a single function can be used across the whole (pT, cosθ∗) range. In each rapidity bin, the signal is ﬁtted using two Gaussians with a com- mon mean, a narrow component fraction f , and a ratio r of broad to narrow widths. These parameters are found to be independent of mass, and are ﬁxed to the average values across the simula- tion samples. The remaining parameter, the width of the narrow component, σ , depends linearly on the mass of the parent state. Together with the splitting functions deﬁned above, this allows the signal shape and the fraction of the signal falling in each of the analysis bins to be determined for any Xb mass. The mass resolution and background shape differ at central and forward rapidities, so the analysis is performed in bins of \|y\| < 1.2 (barrel) and 1.2 < \|y\| < 2.4 (endcap). Several possible ways to ex- ploit the (pT, cosθ∗) discrimination were considered, including fur- ther binning, a diagonal cut in (pT, cosθ∗), and a requirement on the R between the Υ (1S) and each pion (as used by CMS [25]). The choice of method was based on optimising the expected sig- niﬁcance for a weak signal at a mass of 10 561 MeV. In the ﬁnal approach eight analysis bins of varying sensitivity are used, formed from combinations of high and low \|y\|, high and low pT, and high and low cosθ∗. 3. Reconstruction and event selection ATLAS has measured doubly differential pro- duction cross sections for Υ (2S) and Υ (3S) at 7 TeV in pT bins up to 70 GeV for \|y\| < 1.2 and 1.2 < \|y\| < 2.25 [35]. These results can be extended to \|y\| < 2.4 assuming ﬂat rapidity dependence, and up to pT = 100 GeV using CMS measurements [36]. The result- ing Υ (2S) cross section is compared to the production kinematics of a 7 TeV simulation using the 2011 ATLAS tune [37] of Pythia 6.4 [38]. The ratio of the two in (\|y\|, pT) bins deﬁnes production weights, which are applied to the 8 TeV Υ (2S) simulated sample, assuming that Pythia correctly models the increase in cross sec- tion with √ s. The same procedure is used for the Υ (3S); for other masses, linear extrapolation of the Υ (2S) and Υ (3S) weights is used. The simulated Υ (2S) and Υ (3S) →π+π−Υ (1S) samples are further reweighted to match dipion mass distributions observed by CLEO [39,40], to allow comparison of simulation and data. Fig. 1. The π+π−Υ (1S) invariant mass distribution in the kinematic bin most sen- sitive to an Xb signal: \|y\| < 1.2, pT > 20 GeV, and cosθ∗> 0. The only apparent peaks are at the masses of the Υ (2S) (10023 MeV) and Υ (3S) (10355 MeV). as splitting functions. At any speciﬁed mass, the signal yield frac- tion in any particular (\|y\|, pT, cosθ∗) bin can be calculated from an appropriately chosen product of three of these and their comple- ments, (1 −S). For example, the fraction in the bin (\|y\| < 1.2, pT > 20 GeV, cosθ∗< 0) is given by S\|y\| · (1 −Sb pT(m)) · S(3) cos θ∗(m). 5. Fitting strategy The optimal bin boundaries for pT and cosθ∗were determined to be 20 GeV and 0, respectively. Searches for the production of the Υ (10 860) and Υ (11 020), which have natural widths larger than the experimental resolu- tion, are also performed. Each of these states is modelled as a Breit–Wigner convolved with the mass-dependent signal shape de- scribed in the previous paragraph, representing the detector reso- lution. The fractions of the Υ (10 860) signal falling in the eight analysis bins are extracted from the simulation, while those for the Υ (11 020) are determined by extrapolation. All ﬁts presented here are binned, extended maximum-likeli- hood ﬁts in a local region around the mass of interest. The bin width is 2 MeV in all ﬁts. The background is described by a lin- ear combination of Chebychev polynomials up to second-order, with independent parameters in each analysis bin, unless other- wise speciﬁed. The π+π−Υ (1S) mass distribution for the most sensitive bin (\|y\| < 1.2, pT > 20 GeV, cosθ∗> 0) is shown in Fig. 1. The Υ (2S) and Υ (3S) peaks are clearly visible, but no other peaks are ap- parent. The background in this bin decreases with mass above the Υ (3S), whereas the fraction of signal events falling in this bin is constant above the Υ (3S) in the simulation. Thus higher sensitivity is expected at larger masses. 6. Results for the Υ (2S) and Υ (3S) To test kine- matic distributions in the Υ (2S) simulation, signal fractions in the eight analysis bins are checked against their expected values, and are found to be consistent within statistical uncertainties. where the product of the cross section and branching fraction, (σB)2S, is estimated from the extended cross-section measure- ment (see Section 4) using world-average values for the branching fractions B(Υ (1S) →μ+μ−) and B(Υ (2S) →π+π−Υ (1S)) [23]. The shape of the doubly differential cross section is also used to calculate the acceptance, A = (1.442 ± 0.004)%, assuming the CLEO dipion mass spectrum [39] and isotropic signal decays. The reconstruction eﬃciency for decays within the acceptance, ϵ = 0.283 ± 0.002, is taken from the Υ (2S) simulation. To test kine- matic distributions in the Υ (2S) simulation, signal fractions in the eight analysis bins are checked against their expected values, and are found to be consistent within statistical uncertainties. At each mass, the p-value is extracted using the asymptotic formula [41] for the q0 statistic, a modiﬁcation of the standard likelihood ratio (see Fig. 3). No evidence for new states with local signiﬁcance z ≥3 is found. The expected number of Xb events can be written as N = N2S · R · A A2S · ϵ ϵ2S , (2) N = N2S · R · A A2S · ϵ ϵ2S , N = N2S · R · A A2S · ϵ ϵ2S , (2) where R ≡(σB)/(σB)2S, the production rate relative to that of the Υ (2S). The eﬃciency, ϵ, as a function of mass is determined by ﬁtting a function a + b/(1 + e−c(m−d)) to the values from the simulated samples. The acceptance increases with the mass of the parent state due to the increased energy available to the pions and muons. Additionally, the measured production spectra of the Υ (1S), Υ (2S), and Υ (3S) states [35] are progressively harder in pT with increasing mass. The acceptance for a hypothetical Xb state of arbitrary mass is estimated here by linear extrapolation of calculations performed at the Υ (2S) and Υ (3S), using the mea- sured production spectra of these states. The ratio (Aϵ)/(Aϵ)2S rises from 1.1 at m = 10 GeV to 7.5 at m = 11 GeV. 6. Results for the Υ (2S) and Υ (3S) Based on the simulation samples, the fraction of signal in the barrel region \|y\| < 1.2 is independent of mass with an aver- age value of S\|y\| = 0.606 ± 0.004. Within the barrel, the frac- tion of signal with pT < 20 GeV develops smoothly with mass and can be characterised by an analytic turn-on curve Sb pT(m) = a/(1 + e−b(m−c)). In the endcap, the dependence is described by Sec pT(m), which has the same functional form as Sb pT(m) with dif- ferent values for the parameters. Similarly, within each (\|y\|, pT) bin the fraction of the signal with cosθ∗< 0 is modelled with a quadratic function, S(i) cos θ∗(m) = a + bm + cm2, where i = 1–4 labels the bin. These S functions, seven in total, are referred to below Fits to the π+π−Υ (1S) spectrum near the Υ (2S) are ﬁrst performed separately in barrel and endcap bins, across the full (pT, cosθ∗) range, with signal mass and width parameters free in the ﬁt (Figs. 2a and 2b). In both cases, the mass is consistent with the world average for the Υ (2S), and the σ parameters are within uncertainties of the values ﬁtted to the Υ (2S) simulation. Signal shape parameters are then ﬁxed to simulated values to reduce uncertainties, and a separate ﬁt is performed on each anal- ysis bin. The fraction of signal decays falling in the barrel is mea- sured to be 0.67 ± 0.04, compared to 0.606 ± 0.004 in the simula- ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 202 202 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 Fig. 2. The measured π+π−Υ (1S) invariant mass (data points), together with ﬁts (red solid curves) to the Υ (2S) peak in the (a) barrel and (b) endcap, with signal mass (m) and width parameters (σb and σec, respectively) free; and (c) a ﬁt to the Υ (3S) peak in the most sensitive bin, with zero suppressed on the vertical scale. Ns is the ﬁtted signal yield in each bin. The background component (green, long-dashed) and the signal component (peaked blue curve) are also shown separately. In each case, the lower panel shows background-subtracted data, with the total signal function (blue solid) and its two Gaussian components (pink, short-dashed curves) overlaid. 6. Results for the Υ (2S) and Υ (3S) A simultaneous ﬁt to the analysis bins is also performed at the Υ (3S) mass, with reduced χ 2 = 1.0 and statistical signiﬁcance (from the likelihood-ratio test statistic) z = 8.7. An individual ﬁt to the most sensitive bin (shown in Fig. 2(c), with larger binning to emphasise the peak) has signiﬁcance z = 6.5. The total Υ (3S) yield, 11 600 ± 1300, agrees with the prediction estimated analo- gously to Eq. (1), 11 400 ± 1500. 7.1. Hypothesis tests Using Eq. (2) and the formalism from Ref. [41], the expected signiﬁcance for a relative production rate R = 6.56% (the value of the analogous quantity for the X(3872) [5]) is calculated as a func- tion of mass, shown as the dashed blue line in Fig. 3; it exceeds 5σ for m ≳10.12 GeV. Expectations for a weaker signal with R = 3% are also shown (long-dashed red line). Given the null result, up- per limits are calculated on R after modifying the ﬁt to include systematic uncertainties. A hypothesis test for the presence of an Xb peak is performed every 10 MeV from 10 GeV to 11 GeV, assuming a narrow state3 that has a differential cross section with a (\|y\|, pT) distribution similar to that of the Υ (2S) or Υ (3S), decaying according to three- body phase space. The signal shape and bin splittings are treated as described in Section 5. At each mass, a simultaneous ﬁt to the analysis bins is performed in a range m ± 8σec, where σec(m) is the width of the narrow signal component in the endcap: the win- dow varies from ±72 MeV at 10 GeV to ±224 MeV at 10.9 GeV; 3 Here, a narrow state refers to one whose natural width is much smaller than the experimental resolution. 6. Results for the Υ (2S) and Υ (3S) (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 2. The measured π+π−Υ (1S) invariant mass (data points), together with ﬁts (red solid curves) to the Υ (2S) peak in the (a) barrel and (b) endcap, with signal mass (m) and width parameters (σb and σec, respectively) free; and (c) a ﬁt to the Υ (3S) peak in the most sensitive bin, with zero suppressed on the vertical scale. Ns is the ﬁtted signal yield in each bin. The background component (green, long-dashed) and the signal component (peaked blue curve) are also shown separately. In each case, the lower panel shows background-subtracted data, with the total signal function (blue solid) and its two Gaussian components (pink, short-dashed curves) overlaid. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) tion; similar results were seen in 7 TeV data. The ratio 0.67/0.606 is used to rescale the barrel fraction in the simulation. near 11 GeV, m −8σec < m < 11.2 GeV is used. When ﬁtting near the Υ (2S) and Υ (3S) regions, the lineshapes for these signal components are added to the background model, with normali- sations governed by Gaussian constraints to the values obtained from the ﬁts given in Section 6. In the immediate vicinity of the peaks, m2S,3S ± 4σb, no search is performed; the width of the nar- row signal component in the barrel, σb, is 5.66 MeV (9.37 MeV) at the Υ (2S) (Υ (3S)) mass. This reduces the analysis range to 10.05–10.31 and 10.40–11.00 GeV. The total ﬁtted yield N2S = 34 300 ± 800 is consistent with (1) Nexpected 2 = (σB)2S · L · A · ϵ = 33 300 ± 2500, (1) where the product of the cross section and branching fraction, (σB)2S, is estimated from the extended cross-section measure- ment (see Section 4) using world-average values for the branching fractions B(Υ (1S) →μ+μ−) and B(Υ (2S) →π+π−Υ (1S)) [23]. The shape of the doubly differential cross section is also used to calculate the acceptance, A = (1.442 ± 0.004)%, assuming the CLEO dipion mass spectrum [39] and isotropic signal decays. The reconstruction eﬃciency for decays within the acceptance, ϵ = 0.283 ± 0.002, is taken from the Υ (2S) simulation. 7.3. Upper limit calculation Alterna- tive extrapolations between the Υ (1S) and Υ (2S), and between Υ (1S) and Υ (3S), are also tried; the greatest change in the accep- tance ratio, 12%, is assigned as the uncertainty. If an Xb state exists and lies within the range of masses to which this analysis is sensitive, its production cross section and/or its branching fraction must be lower, relative to the Υ (2S), than that of the X(3872) relative to the ψ(2S). There are arguments that the decay Xb →π+π−Υ (1S) should be suppressed, in the absence of the strong isospin-violating effects that are present for X(3872) →π+π−J/ψ [46,47]. In this case the Xb would have more prominent decays to π+π−χb1, π+π−π 0Υ (1S), and other ﬁnal states which are relatively diﬃcult to reconstruct. The parameters of the eﬃciency, the splitting functions, and the widths of the narrow signal components σb and σec as functions of mass, are varied by the uncertainties on their ﬁtted values; al- ternative functional forms are also tried. In each case, the largest deviation is assigned as the systematic uncertainty. The use of production weights (described in Section 4) relies on assumptions regarding rapidity dependence, and evolution from √ s = 7 TeV to 8 TeV. Removing these weights produces a ∼1% change in eﬃ- ciency ratio (most of the differences cancel), but changes the val- ues of the splitting functions by up to 8%. All results to this point assume that any hypothetical Xb pro- duction is unpolarised. Angular distributions of the Υ (1S, 2S, 3S) states in pp collisions are consistent with unpolarised produc- tion [31], but the Xb spin-alignment is unknown and can have a strong impact on the eﬃciency ratio, acceptance ratio, and bin splitting fractions. Rather than including this as a systematic un- certainty, upper limits are recalculated under longitudinal (‘LONG’) and three transverse (‘TRPP’, ‘TRP0’, ‘TRPM’) spin-alignment sce- narios [48]. Shifts in the upper limits (either up or down) depend only weakly on mass; the shift is smaller at large masses. In Fig. 4 the effect of each hypothesis is represented by a single number, Data versus simulation differences in the Υ (2S) width param- eters in the barrel and endcap (1.9% and 4.2%, respectively) are incorporated as a source of uncertainty, as is the statistical uncer- tainty on the averages used for signal shape parameters f and r (0.5–1.4%). 7.3. Upper limit calculation falling in each of the analysis bins. From Eq. (2), the upper limit on R is proportional to the inverse ﬁtted Υ (2S) yield, N−1 2S , and the ratios A2S/A and ϵ2S/ϵ. For each source of systematic uncertainty, the impact on these factors is quantiﬁed to ﬁnd the maximum shift across the mass range. These are then summed in quadrature and included in the ﬁt as Gaussian-constrained nuisance parameters. Upper limits are evaluated at the 95% conﬁdence level using the CLS method by implementing asymptotic formulae for the ˜qμ statistic [41]. The results (Fig. 4, solid line) range between R = 0.8% and 4.0%. Median expected upper limits assuming background only (dashed line), and corresponding ±1σ and ±2σ bands are also shown. These limits include the effect of systematic uncertainties: their inclusion increased the observed limits by up to 13% and in- ﬂated the ±1σ band by 9.5–25%, depending on the Xb mass. The X(3872) →π+π−J/ψ dipion mass distribution favours high mass [6,9]; for a potential hidden-beauty counterpart this distribution is unknown. For ψ(2S) →π+π−J/ψ [42], and both Υ (2S) [39] and Υ (4S) →π+π−Υ (1S) [43,44], the dipion mass distributions are concentrated near the upper boundary; those for Y(4260) →π+π−J/ψ [45] and Υ (3S) →π+π−Υ (1S) [40] are double-humped. The results quoted here assume decay according to three-body phase space; Υ (2S)- and Υ (3S)-like distributions change the splitting functions by up to 35%, decrease the eﬃciency ratio by up to 17%, and produce modest changes in other parame- ters. As a check, upper limits are recalculated with modiﬁed ﬁtting ranges (m ± 7σec and m ± 9σec) and doubled bin widths in the π+π−Υ (1S) mass distributions: shifts are small compared to the ±1σ bands. If an Υ (2S)-like mπ+π−distribution is assumed (cf. CMS [25]), expected upper limits increase: the fractional change is +17% at 10.1 GeV, and ∼+5% for m > 10.4 GeV. These results exclude Xb states with R = 6.56% for masses 10.05–10.31 GeV and 10.40–11.00 GeV. The expected upper limits are more restrictive than those from CMS above m ∼10.1 GeV, and improve as a function of mass; the discrimination in (pT, cosθ∗), exploited by the binning method, becomes increasingly important as mass increases. The next largest contribution is due to the linear extrapolation of the acceptance between the Υ (2S) and Υ (3S) values. 7.2. Systematic uncertainties The upper limit calculation depends indirectly on signal and background ﬁtting parameters, including the fraction of the signal ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 203 ATLAS Collaboration / Physi Fig. 3. The solid curve shows the observed local p-value for the background-only hy- pothesis (left scale), and the corresponding signiﬁcance, z, of a peak in π+π−Υ (1S) (right scale), as a function of the mass of a hypothetical Xb parent state. Also shown are the expected values for the case of a signal with relative production rates (σB)/(σB)2S of 3% (red, long-dashed) and 6.56% (blue, dashed curve). Fig. 4. Observed 95% CLS upper limits (solid line) on the relative production rate R = (σB)/(σB)2S of a hypothetical Xb parent state decaying isotropically to π+π−Υ (1S), as a function of mass. The median expectation (dashed) and the cor- responding ±1σ and ±2σ bands (green and yellow respectively) are also shown. The bar on the right shows typical shifts under alternative Xb spin-alignment sce- narios, relative to the isotropic (‘FLAT’) case shown with the solid point. Fig. 3. The solid curve shows the observed local p-value for the background-only hy- pothesis (left scale), and the corresponding signiﬁcance, z, of a peak in π+π−Υ (1S) (right scale), as a function of the mass of a hypothetical Xb parent state. Also shown are the expected values for the case of a signal with relative production rates (σB)/(σB)2S of 3% (red, long-dashed) and 6.56% (blue, dashed curve). Fig. 4. Observed 95% CLS upper limits (solid line) on the relative production rate R = (σB)/(σB)2S of a hypothetical Xb parent state decaying isotropically to π+π−Υ (1S), as a function of mass. The median expectation (dashed) and the cor- responding ±1σ and ±2σ bands (green and yellow respectively) are also shown. The bar on the right shows typical shifts under alternative Xb spin-alignment sce- narios, relative to the isotropic (‘FLAT’) case shown with the solid point. Fig. 4. Observed 95% CLS upper limits (solid line) on the relative production rate R = (σB)/(σB)2S of a hypothetical Xb parent state decaying isotropically to π+π−Υ (1S), as a function of mass. The median expectation (dashed) and the cor- responding ±1σ and ±2σ bands (green and yellow respectively) are also shown. The bar on the right shows typical shifts under alternative Xb spin-alignment sce- narios, relative to the isotropic (‘FLAT’) case shown with the solid point. Fig. 7.2. Systematic uncertainties 3. The solid curve shows the observed local p-value for the background-only hy- pothesis (left scale), and the corresponding signiﬁcance, z, of a peak in π+π−Υ (1S) (right scale), as a function of the mass of a hypothetical Xb parent state. Also shown are the expected values for the case of a signal with relative production rates (σB)/(σB)2S of 3% (red, long-dashed) and 6.56% (blue, dashed curve). 8. Results for the Υ (13D J ) triplet, Υ (10 860), and Υ (11 020) The search described above does not account for the closely spaced Υ (13D J) triplet or the broad Υ (10 860) and Υ (11 020). To ﬁt for the Υ (13D J), two extra peaks are added to the sig- nal model. CLEO [18] and BaBar [19] have measured the Υ (13D2) mass, with an average of (10 163.7 ± 1.4) MeV, but the mass split- ting within the triplet is unknown. Averaging over several mod- els [49] leads to triplet masses 10 156, 10 164, and 10 170 MeV (at 1 MeV precision). A ﬁt is performed using these values, as- suming independent normalisations but common signal shapes and bin splitting fractions. A signiﬁcance of z = 0.12 is found, with ﬁtted yields −1000 ± 3100, 600 ± 1800, and 800 ± 2300 for J = 1, 2, and 3. Reasonable changes to the mass splittings do not appreciably increase the signiﬁcance, so there appears to be no ev- idence for Υ (13D J) production. Assuming that J = 2 production dominates, or that the mass splitting is larger than the experi- mental resolution, the upper limit on R can be read from Fig. 4; combined with the measured Υ (13D2) →π+π−Υ (1S) branching fraction [19], this yields an upper limit on the relative cross section σ(pp →Υ (13D2))/σ(pp →Υ (2S)) ≤0.55. The crucial computing support from all WLCG partners is ac- knowledged gratefully, in particular from CERN and the ATLAS Tier- 1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Swe- den), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA) and in the Tier-2 facilities worldwide. (pp ( ))/ (pp ( )) The signal model for Υ (10 860) and Υ (11 020) is described in Section 5. Due to the large natural widths of these states, the ﬁtting range is extended to 10.498–11.198 GeV and the back- ground polynomial order increased to three. Signiﬁcances z = 0.6 and z = 0.3 are found for Υ (10 860) and Υ (11 020), for masses and widths ﬁxed to world-average values [23]. As these parame- ters have large uncertainties, the signiﬁcance is also calculated in a grid of m ± 20 MeV and Γ ± Γ , where Γ is the uncertainty on the world-average width [23]. 9. Conclusions A search for a hidden-beauty analogue of the X(3872) is conducted by reconstructing π+π−Υ (1S)(→μ+μ−) events in 16.2 fb−1 of pp collision data recorded at √ s = 8 TeV by ATLAS at the LHC. To optimise the sensitivity of the search, the analysis is performed in eight bins of rapidity, transverse momentum, and the angle (in the rest frame of the parent state) between the di- pion system and the laboratory-frame momentum of the parent. At each mass, the presence of a signal is tested by performing simultaneous ﬁts to the nearby π+π−Υ (1S) mass spectrum in these bins; no evidence for new narrow states is found for masses 10.05–10.31 GeV and 10.40–11.00 GeV. Upper limits are also set on the ratio R = [σ(pp →Xb)B(Xb →π+π−Υ (1S))]/[σ(pp → Υ (2S))B(Υ (2S) →π+π−Υ (1S))], with results ranging from 0.8% to 4.0% depending on the Xb mass. The analogous ratio for the X(3872) is 6.56%: a value this large is excluded for all Xb masses considered. Separate ﬁts to the Υ (13D J) triplet, Υ (10 860), and Υ (11 020) also reveal no signiﬁcant signals, and a CLS upper limit of 0.55 is set on σ(pp →Υ (13D2))/σ(pp →Υ (2S)). [5] CMS Collaboration, Measurement of the X(3872) production cross section via decays to J/ψπ+π−in pp collisions at √ s = 7 TeV, J. High Energy Phys. 1304 (2013) 154, http://dx.doi.org/10.1007/JHEP04(2013)154, arXiv:1302.3968. [6] A. Abulencia, et al., CDF Collaboration, Measurement of the di-pion mass spec- trum in X(3872) →J/ψπ+π−decays, Phys. Rev. Lett. 96 (2006) 102002, http://dx.doi.org/10.1103/PhysRevLett.96.102002, arXiv:hep-ex/0512074. [7] B. Aubert, et al., BaBar Collaboration, Search for B+ →X(3872)K +, X(3872) →J/ψγ , Phys. Rev. D 74 (2006) 071101, http://dx.doi.org/10.1103/ PhysRevD.74.071101, arXiv:hep-ex/0607050. J/ψγ y ( ) PhysRevD.74.071101, arXiv:hep-ex/0607050. [8] A. Abulencia, et al., CDF Collaboration, Analysis of the quantum numbers J PC of the X(3872), Phys. Rev. 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Finally, uncertainties on N2S and the bar- rel/endcap scaling factor are assigned based on uncertainties from the Υ (2S) ﬁts. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 204 chosen as the maximum difference in the median expected signif- icance from the unpolarised (‘FLAT’) case. chosen as the maximum difference in the median expected signif- icance from the unpolarised (‘FLAT’) case. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azer- baijan; 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, DNSRC and Lundbeck Foundation, Den- mark; EPLANET, ERC and NSRF, European Union; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, DFG, HGF, MPG and AvH Foundation, Germany; GSRT and NSRF, Greece; ISF, MINERVA, GIF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; BRF and RCN, Norway; MNiSW and NCN, Poland; GRICES and FCT, Portugal; MNE/IFA, Romania; MES of Russia and ROSATOM, Rus- sian Federation; JINR; MSTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SER, SNSF and Cantons of Bern and Geneva, Switzerland; NSC, Taiwan; TAEK, Turkey; STFC, the Royal Society and Leverhulme Trust, United Kingdom; DOE and NSF, United States of America. 9. 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Schouten 160a, J. Schovancova 25, S. Schramm 159, M. Schreyer 175, C. Schroeder 82, N. Schuh 82, M.J. Schultens 21, H.-C. Schultz-Coulon 58a, H. Schulz 16, M. Schumacher 48, B.A. Schumm 138, Ph. Schune 137, C. Schwanenberger 83, A. Schwartzman 144, T.A. Schwarz 88, Ph. Schwegler 100, Ph. Schwemling 137, R. Schwienhorst 89, J. Schwindling 137, T. Schwindt 21, M. Schwoerer 5, F.G. Sciacca 17, E. Scifo 116, G. Sciolla 23, W.G. Scott 130, F. Scuri 123a,123b, F. Scutti 21, J. Searcy 88, G. Sedov 42, E. Sedykh 122, S.C. Seidel 104, A. Seiden 138, F. Seifert 127, J.M. Seixas 24a, G. Sekhniaidze 103a, S.J. Sekula 40, K.E. Selbach 46, D.M. Seliverstov 122,∗, G. Sellers 73, N. Semprini-Cesari 20a,20b, C. Serfon 30, L. Serin 116, L. Serkin 54, T. Serre 84, R. Seuster 160a, H. Severini 112, T. Sﬁligoj 74, F. Sforza 100, A. Sfyrla 30, E. Shabalina 54, M. Shamim 115, L.Y. Shan 33a, R. Shang 166, J.T. Shank 22, M. Shapiro 15, P.B. Shatalov 96, K. Shaw 165a,165b, C.Y. Shehu 150, P. Sherwood 77, L. Shi 152,ae, S. Shimizu 66, C.O. Shimmin 164, M. Shimojima 101, M. Shiyakova 64, A. Shmeleva 95, M.J. Shochet 31, D. Short 119, S. Shrestha 63, E. Shulga 97, M.A. Shupe 7, S. Shushkevich 42, P. Sicho 126, O. Sidiropoulou 155, D. Sidorov 113, A. Sidoti 133a, F. Siegert 44, Dj. Sijacki 13a, J. Silva 125a,125d, Y. Silver 154, D. Silverstein 144, S.B. Silverstein 147a, V. Simak 127, O. Simard 5, Lj. Simic 13a, S. Simion 116, E. Simioni 82, B. Simmons 77, R. Simoniello 90a,90b, M. Simonyan 36, P. Sinervo 159, N.B. Sinev 115, V. Sipica 142, G. Siragusa 175, A. Sircar 78, A.N. Sisakyan 64,∗, S.Yu. Sivoklokov 98, J. Sjölin 147a,147b, T.B. Sjursen 14, H.P. Skottowe 57, K.Yu. Skovpen 108, P. Skubic 112, M. Slater 18, T. Slavicek 127, K. Sliwa 162, V. Smakhtin 173, B.H. Smart 46, L. Smestad 14, S.Yu. Smirnov 97, Y. Smirnov 97, L.N. Smirnova 98,af , O. Smirnova 80, K.M. Smith 53, M. Smizanska 71, K. Smolek 127, A.A. Snesarev 95, G. Snidero 75, S. Snyder 25, R. Sobie 170,j, F. Socher 44, A. Soffer 154, D.A. Soh 152,ae, C.A. Solans 30, M. Solar 127, J. Solc 127, E.Yu. Soldatov 97, U. Soldevila 168, A.A. Solodkov 129, A. Soloshenko 64, O.V. Solovyanov 129, V. ATLAS Collaboration Solovyev 122, P. Sommer 48, H.Y. Song 33b, N. Soni 1, A. Sood 15, A. Sopczak 127, B. Sopko 127, V. Sopko 127, V. Sorin 12, M. Sosebee 8, R. Soualah 165a,165c, P. Soueid 94, A.M. Soukharev 108,c, D. South 42, S. Spagnolo 72a,72b, F. Spanò 76, W.R. Spearman 57, F. Spettel 100, R. Spighi 20a, G. Spigo 30, L.A. Spiller 87, M. Spousta 128, T. Spreitzer 159, B. Spurlock 8, R.D. St. Denis 53,∗, S. Staerz 44, J. Stahlman 121, R. Stamen 58a, S. Stamm 16, E. Stanecka 39, R.W. Stanek 6, C. Stanescu 135a, M. Stanescu-Bellu 42, M.M. Stanitzki 42, S. Stapnes 118, E.A. Starchenko 129, J. Stark 55, P. Staroba 126, P. Starovoitov 42, R. Staszewski 39, P. Stavina 145a,∗, P. Steinberg 25, B. Stelzer 143, H.J. Stelzer 30, O. Stelzer-Chilton 160a, H. Stenzel 52, S. Stern 100, G.A. Stewart 53, J.A. Stillings 21, J. Rieger 54, M. Rijssenbeek 149, A. Rimoldi 120a,120b, L. Rinaldi 20a, E. Ritsch 61, I. Riu 12, F. Rizatdinova 113, . Rizvi 75, S.H. Robertson 86,j, A. Robichaud-Veronneau 86, D. Robinson 28, J.E.M. Ro b . Robson 53, C. Roda 123a,123b, L. Rodrigues 30, S. Roe 30, O. Røhne 118, S. Rolli 162, A , , , , , E. Rossi 103a,103b, L.P. Rossi 50a, R. Rosten 139, M. Rotaru 26a, I. Roth 173, J. Rothber J C.R. Royon 137, A. Rozanov 84, Y. Rozen 153, X. Ruan 146c, F. Rubbo 12, I. Rubinskiy 44 159 48 30 48 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 213 K. Suruliz 150, G. Susinno 37a,37b, M.R. Sutton 150, Y. Suzuki 65, M. Svatos 126, S. Swedish 169, M. Swiatlowski 144, I. Sykora 145a, T. Sykora 128, D. Ta 89, C. Taccini 135a,135b, K. Tackmann 42, J. Taenzer 159, A. Taffard 164, R. Taﬁrout 160a, N. Taiblum 154, H. Takai 25, R. Takashima 68, H. Takeda 66, T. Takeshita 141, Y Takubo 65 M Talby 84 A A Talyshev 108,c J Y C Tam 175 K G Tan 87 J Tanaka 156 R Tanaka 116 J. Therhaag 21, T. Theveneaux-Pelzer 34, J.P. Thomas 18, J. Thomas-Wilsker 76, E.N. Thompson 35, , , y y , , , , S.A. Tupputi 20a,20b, S. Turchikhin 98,af , D. Turecek 127, I. Turk Cakir 4d, R. Turra 90a,90b, P.M. Tuts 35, A. Tykhonov 49, M. Tylmad 147a,147b, M. Tyndel 130, K. Uchida 21, I. ATLAS Collaboration Ueda 156, R. Ueno 29, M. Ughetto 84, M. Ugland 14, M. Uhlenbrock 21, F. Ukegawa 161, G. Unal 30, A. Undrus 25, G. Unel 164, F.C. Ungaro 48, Y. Unno 65, C. Unverdorben 99, D. Urbaniec 35, P. Urquijo 87, G. Usai 8, A. Usanova 61, L. Vacavant 84, V. Vacek 127, B. Vachon 86, N. Valencic 106, S. Valentinetti 20a,20b, A. Valero 168, L. Valery 34, S. Valkar 128, E. Valladolid Gallego 168, S. Vallecorsa 49, J.A. Valls Ferrer 168, W. Van Den Wollenberg 106, ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 214 C. Weiser 48, H. Weits 106, P.S. Wells 30, T. Wenaus 25, D. Wendland 16, Z. Weng 152,ae, T. Wengler 30, S. Wenig 30, N. Wermes 21, M. Werner 48, P. Werner 30, M. Wessels 58a, J. Wetter 162, K. Whalen 29, A. White 8, M.J. White 1, R. White 32b, S. White 123a,123b, D. Whiteson 164, D. Wicke 176, F.J. Wickens 130, W. Wiedenmann 174, M. Wielers 130, P. Wienemann 21, C. Wiglesworth 36, L.A.M. Wiik-Fuchs 21, P.A. Wijeratne 77, A. Wildauer 100, M.A. Wildt 42,ak, H.G. Wilkens 30, J.Z. Will 99, H.H. Williams 121, S. Williams 28, C. Willis 89, S. Willocq 85, A. Wilson 88, J.A. Wilson 18, I. Wingerter-Seez 5, F. Winklmeier 115, B.T. Winter 21, M. Wittgen 144, T. Wittig 43, J. Wittkowski 99, S.J. Wollstadt 82, M.W. Wolter 39, H. Wolters 125a,125c, B.K. Wosiek 39, J. Wotschack 30, M.J. Woudstra 83, K.W. Wozniak 39, M. Wright 53, M. Wu 55, S.L. Wu 174, X. Wu 49, Y. Wu 88, E. Wulf 35, T.R. Wyatt 83, B.M. Wynne 46, S. Xella 36, M. Xiao 137, D. Xu 33a, L. Xu 33b,al, B. Yabsley 151, S. Yacoob 146b,am, R. Yakabe 66, M. Yamada 65, H. Yamaguchi 156, Y. Yamaguchi 117, A. Yamamoto 65, K. Yamamoto 63, S. Yamamoto 156, T. Yamamura 156, T. Yamanaka 156, K. Yamauchi 102, Y. Yamazaki 66, Z. Yan 22, H. Yang 33e, H. Yang 174, U.K. Yang 83, Y. Yang 110, S. Yanush 92, L. Yao 33a, W-M. Yao 15, Y. Yasu 65, E. Yatsenko 42, K.H. Yau Wong 21, J. Ye 40, S. Ye 25, I. Yeletskikh 64, A.L. Yen 57, E. Yildirim 42, M. Yilmaz 4b, R. Yoosoofmiya 124, K. Yorita 172, R. Yoshida 6, K. Yoshihara 156, C. Young 144, C.J.S. Young 30, S. Youssef 22, D.R. Yu 15, J. Turkey 21 Physikalisches Institut, University of Bonn, Bonn, Germany 22 Department of Physics, Boston University, Boston, MA, United States 22 Department of Physics, Boston University, Boston, MA, United States 3 Department of Physics, Brandeis University, Waltham, MA, United Stat p f y , y, , , 24 (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 25 p f y y 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (d) 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department 24 (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 4 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; J / / J p Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Univers ysics Department, Brookhaven National Laboratory, Upton, NY, United St Physics Department, Brookhaven National Laboratory, Upton, NY, United States 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 27 onal Laboratory, Upton, NY, United States uclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, ucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania y p , y, p , , 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania , y, p , , nstitute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technolog University Politehnica Bucharest Bucharest; (d) West University in Timisoara Timisoara Romania 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and M ( ) (d) 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research a 6 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b f y g g, ; f p f p g , y p , Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania luj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West Un Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 7 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Ar 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 29 29 Department of Physics, Carleton University, Ottawa, ON, Canada 30 CERN, Geneva, Switzerland ATLAS Collaboration Yang 174, U.K. Yang 83, Y. Yang 110, S. Yanush 92, L. Yao 33a, W-M. Yao 15, Y. Yasu 65, E. Yatsenko 42, K.H. Yau Wong 21, J. Ye 40, S. Ye 25, I. Yeletskikh 64, A.L. Yen 57, E. Yildirim 42, M. Yilmaz 4b, R. Yoosoofmiya 124, K. Yorita 172, R. Yoshida 6, K. Yoshihara 156, C. Young 144, C.J.S. Young 30, S. Youssef 22, D.R. Yu 15, J. Yu 8, J.M. Yu 88, J. Yu 113, L. Yuan 66, A. Yurkewicz 107, I. Yusuff 28,an, B. Zabinski 39, R. Zaidan 62, A.M. Zaitsev 129,aa, A. Zaman 149, S. Zambito 23, L. Zanello 133a,133b, D. Zanzi 87, C. Zeitnitz 176, M. Zeman 127, A. Zemla 38a, K. Zengel 23, O. Zenin 129, T. Ženiš 145a, D. Zerwas 116, G. Zevi della Porta 57, D. Zhang 88, F. Zhang 174, H. Zhang 89, J. Zhang 6, L. Zhang 152, X. Zhang 33d, Z. Zhang 116, Z. Zhao 33b, A. Zhemchugov 64, J. Zhong 119, B. Zhou 88, L. Zhou 35, N. Zhou 164, C.G. Zhu 33d, H. Zhu 33a, J. Zhu 88, Y. Zhu 33b, X. Zhuang 33a K. Zhukov 95, A. Zibell 175, D. Zieminska 60, N.I. Zimine 64, C. Zimmermann 82, R. Zimmermann 21, S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 142, G. Zobernig 174, A. Zoccoli 20a,20b, M. zur Nedden 16, G. Zurzolo 103a,103b, V. Zutshi 107, L. Zwalinski 30 S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 142, G. Zobernig 174, A. Zoccoli 20a,20b, M. zur Nedden 16, G. Zurzolo 103a,103b, V. Zutshi 107, L. ATLAS Collaboration Zwalinski 30 1 Department of Physics, University of Adelaide, Adelaide, Australia 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany, NY, United States 3 Department of Physics, University of Alberta, Edmonton, AB, Canada , , , versity, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara ara, Turkey p f y , y f , , , 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d) Turkish Atomic Energy Authority, Ankara, Turkey p f y , y f , , , 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d) Turkish Atomic Energy Authority, Ankara, Turkey 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, T (d) Turkish Atomic Energy Authority, Ankara, Turkey 4 (a) Department of Physics, Ankara University, Ankara; (b) Departmen (d) Turkish Atomic Energy Authority, Ankara, Turkey (d) Turkish Atomic Energy Authority, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annec 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United State 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United States 7 Department of Physics, University of Arizona, Tucson, AZ, United Stat 7 Department of Physics, University of Arizona, Tucson, AZ, United States p f y y f 8 Department of Physics, The University of Texas at Arlington, Arlington, TX, United States 8 Department of Physics, The University of Texas at Arlington, Arlington, TX, United States 8 Department of Physics, The University of Texas at Arlington, 9 Physics Department, University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zograf 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan f y j y f j 12 Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona, Barcelona, Spain b 12 Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona, B 12 Institut de Física d’Altes Energies and Departament de Física de la U g p 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, U 3 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Insti 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 14 Department for Physics and Technology, University of Bergen, Bergen, Norway 14 Department for Physics and Technology, University of Bergen, Bergen, Norway 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States 15 Physics Division, Lawrence Berkeley National Laboratory and U 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States 16 Department of Physics, Humboldt University, Berlin, Germany 16 Department of Physics, Humboldt University, Berlin, Germany 16 Department of Physics, Humboldt University, Berlin, Germany 17 p f y y y 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18 17 Albert Einstein Center for Fundamental Physics and Laboratory 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, Univ 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics E Turkey 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey ATLAS Collaboration Yu 8, J.M. Yu 88, J. Yu 113, L. Yuan 66, A. Yurkewicz 107, I. Yusuff 28,an, B. Zabinski 39, R. Zaidan 62, A.M. Zaitsev 129,aa, A. Zaman 149, S. Zambito 23, L. Zanello 133a,133b, D. Zanzi 87, C. Zeitnitz 176, M. Zeman 127, A. Zemla 38a, K. Zengel 23, O. Zenin 129, T. Ženiš 145a, D. Zerwas 116, G. Zevi della Porta 57, D. Zhang 88, F. Zhang 174, H. Zhang 89, J. Zhang 6, L. Zhang 152, X. Zhang 33d, Z. Zhang 116, Z. Zhao 33b, A. Zhemchugov 64, J. Zhong 119, B. Zhou 88, L. Zhou 35, N. Zhou 164, C.G. Zhu 33d, H. Zhu 33a, J. Zhu 88, Y. Zhu 33b, X. Zhuang 33a, K. Zhukov 95, A. Zibell 175, D. Zieminska 60, N.I. Zimine 64, C. Zimmermann 82, R. Zimmermann 21, S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 142, G. Zobernig 174, A. Zoccoli 20a,20b, M. zur Nedden 16, G. Zurzolo 103a,103b, V. Zutshi 107, L. Zwalinski 30 C. Weiser 48, H. Weits 106, P.S. Wells 30, T. Wenaus 25, D. Wendland 16, Z. Weng 152,ae, T. Wengler 30, S. Wenig 30, N. Wermes 21, M. Werner 48, P. Werner 30, M. Wessels 58a, J. Wetter 162, K. Whalen 29, A. White 8, M.J. White 1, R. White 32b, S. White 123a,123b, D. Whiteson 164, D. Wicke 176, F.J. Wickens 130, W. Wiedenmann 174, M. Wielers 130, P. Wienemann 21, C. Wiglesworth 36, L.A.M. Wiik-Fuchs 21, P.A. Wijeratne 77, A. Wildauer 100, M.A. Wildt 42,ak, H.G. Wilkens 30, J.Z. Will 99, H.H. Williams 121, S. Williams 28, C. Willis 89, S. Willocq 85, A. Wilson 88, J.A. Wilson 18, I. Wingerter-Seez 5, F. Winklmeier 115, B.T. Winter 21, M. Wittgen 144, T. Wittig 43, J. Wittkowski 99, S.J. Wollstadt 82, M.W. Wolter 39, H. Wolters 125a,125c, B.K. Wosiek 39, J. Wotschack 30, M.J. Woudstra 83, K.W. Wozniak 39, M. Wright 53, M. Wu 55, S.L. Wu 174, X. Wu 49, Y. Wu 88, E. Wulf 35, T.R. Wyatt 83, B.M. Wynne 46, S. Xella 36, M. Xiao 137, D. Xu 33a, L. Xu 33b,al, B. Yabsley 151, S. Yacoob 146b,am, R. Yakabe 66, M. Yamada 65, H. Yamaguchi 156, Y. Yamaguchi 117, A. Yamamoto 65, K. Yamamoto 63, S. Yamamoto 156, T. Yamamura 156, T. Yamanaka 156, K. Yamauchi 102, Y. Yamazaki 66, Z. Yan 22, H. Yang 33e, H. 30 CERN, Geneva, Switzerland Andronikashvili Institute of Physics, Iv. 30 CERN, Geneva, Switzerland Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 91 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, M 92 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belarus 92 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belarus 93 Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, United States Department of Physics, Massachusetts Institute of Technology, Cambridg 94 Group of Particle Physics, University of Montreal, Montreal, QC, Canad 94 Group of Particle Physics, University of Montreal, Montreal, QC, Cana 95 P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 96 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia f p y ( ) 97 National Research Nuclear University MEPhI, Moscow, Russia 97 National Research Nuclear University MEPhI, Moscow, Russia 97 National Research Nuclear University MEPhI, Moscow, Russia 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov M 100 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 100 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, G 100 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), Münc 101 Nagasaki Institute of Applied Science, Nagasaki, Japan 101 Nagasaki Institute of Applied Science, Nagasaki, Japan f y g y y g y J p 103 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy f y g y y g y J p 103 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy 104 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, United States 106 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherla 106 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 107 Department of Physics, Northern Illinois University, DeKalb, IL, United States 107 Department of Physics, Northern Illinois University, DeKalb, IL, United States 108 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 108 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 109 Department of Physics, New York University, New York, NY, United States 109 Department of Physics, New York University, New York, NY, United States 110 Ohio State University, Columbus, OH, United States 111 Faculty of Science, Okayama University, Okayama, Japan y f y y y J p 112 Homer L. 30 CERN, Geneva, Switzerland 31 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States , y f g , g , , 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Department, Shanghai Jiao Tong University, Shanghai, China 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 35 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of d 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 ( ) (b) ( ) 2 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Sa p , ﬁ , g ; p , 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Department, Shanghai Jiao Tong University, Shanghai, China 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 35 hysics, Nanjing University, Jiangsu; (d) School of Physics, Shandong Univ Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Departme y j g y J g f y g y g y p g J g y g 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 35 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Fe 35 34 Laboratoire de Physique Corpusculaire, Clermont Université and Uni 35 Nevis Laboratory, Columbia University, Irvington, NY, United States 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fisica, Università della Calabria, Rende, Italy 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fisica, Università della Calabria, Rende 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fis 7 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 215 nology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, 38 (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 Poland 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 40 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Po y f y y f 40 Physics Department, Southern Methodist University, Dallas, TX, United States 40 Physics Department, Southern Methodist University, Dallas, TX, United States y p y 41 Physics Department, University of Texas at Dallas, Richardson, TX, Un y p y Physics Department, University of Texas at Dallas, Richardson, TX, Unite 42 DESY, Hamburg and Zeuthen, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortm 44 Institut für Kern- und Teilchenphysik, Technische Universität Dresd 45 Department of Physics, Duke University, Durham, NC, United States 45 Department of Physics, Duke University, Durham, NC, United States 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46 SUPA – School of Physics and Astronomy, University of Edinburgh, E 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United King 47 INFN Laboratori Nazionali di Frascati, Frascati, Italy 48 Fakultät für Mathematik und Physik, Albert-Ludwigs 48 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 49 Section de Physique, Université de Genève, Geneva, Switzerland 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università 51 (a) E. 30 CERN, Geneva, Switzerland Javakhishvili Tbilisi State University, Tbilisi; (b) f y , J y, ; g gy y , y, , g 52 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53 ikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53 SUPA – School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdo 54 II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France Laboratoire de Physique Subatomique et de Cosmologie, Université Gren y q q g 56 Department of Physics, Hampton University, Hampton, VA, United States 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, MA, United States b 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, MA, United States Laboratory for Particle Physics and Cosmology, Harvard University, Cam hysics and Cosmology, Harvard University, Cambridge, MA, United States Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für ht K l U i ität H id lb M h i G 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisch 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Hei 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiros 60 Department of Physics, Indiana University, Bloomington, IN, United St 60 Department of Physics, Indiana University, Bloomington, IN, United States p f y y g 61 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, In 61 Institut für Astro- und Teilchenphysik, Leopold-Fr 61 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austr 62 University of Iowa, Iowa City, IA, United States 63 Department of Physics and Astronomy, Iowa State University, Ame 63 Department of Physics and Astronomy, Iowa State University, Ames, IA, United State 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 65 KEK, High Energy Accelerator Research Organization, Tsukuba, J 66 Graduate School of Science, Kobe University, Kobe, J 67 Faculty of Science, Kyoto University, Kyoto, Japan 68 Kyoto University of Education, Kyoto, Japan y y f y J p 69 Department of Physics, Kyushu University, Fukuoka, Japan 69 Department of Physics, Kyushu University, Fukuoka, Japan 70 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 70 Instituto de Física La Plata, Universidad Nacional de La Plata 70 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 71 71 Physics Department, Lancaster University, Lancaster, United Kingdom y p y g 72 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 72 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Ita 72 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Un 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 74 Department of Physics, Jožef Stefan Institute and University of Ljublja 75 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdo 75 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom f y y y y f g 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 77 Department of Physics and Astronomy, University C 78 Louisiana Tech University, Ruston, LA, United States 79 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 80 79 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris- 80 Fysiska institutionen, Lunds universitet, Lund, Sweden y 81 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 82 81 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 81 Departamento de Fisica Teorica C-15, Universidad Aut 82 Institut für Physik, Universität Mainz, Mainz, German 83 School of Physics and Astronomy, University of Manchester, Manch 84 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 84 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 85 Department of Physics, University of Massachusetts, Amherst, MA, United Sta 85 Department of Physics, University of Massachusetts, Amherst, MA 86 Department of Physics, McGill University, Montreal, QC, Canada 87 School of Physics, University of Melbourne, Victoria, Australia 87 School of Physics, University of Melbourne, Victoria, Australia 88 Department of Physics, The University of Michigan, Ann Arbor, MI, United Stat 88 Department of Physics, The University of Michigan, Ann Arbor, 89 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United S 89 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United States p f y y g y g 90 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 90 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy p y 91 B.I. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 Fermi, Università di Pisa, Pisa, Italy p y 124 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United St 124 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States 125 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa; (b) Faculd p f y y, y f g , g , , 125 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa; (b) Faculdade de Ciências, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (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 Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 126 I tit t f Ph i A d f S i f th C h R bli P h C h R bli Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP Laboratorio de Instrumentacao e Fisica Experimental de Particulas LIP, Lisboa; Faculdade de Ciências, Universidade de Lisboa, Lisb Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; ( ) p Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minh ( ) bra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisb Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de , ; , ; p , , g ; p y Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 126 p g p y Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade d Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep 126 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 126 Institute of Physics, Academy of Sciences of the Czech Republic, 127 Czech Technical University in Prague, Praha, Czech Republic 128 Faculty of Mathematics and Physics, Charles University in Prague, P y f y y g 129 State Research Center Institute for High Energy Physics, Protvino, Russia 130 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 130 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 130 Particle Physics Department, Rutherford Appleton Laboratory, Di y p , f pp y, , 131 Physics Department, University of Regina, Regina, SK, Canada 131 Physics Department, University of Regina, Regina, SK, Canada 132 Ritsumeikan University, Kusatsu, Shiga, Japan 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 133 (a) INFN Sezione di Roma; (b) Dipartimento di F 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Univ 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy p y 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 – Agdal Rabat Morocco 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre Na 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassa Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies Université Hassan II, Casablanca; 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 Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Universi Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA – Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda sciences, Université Mohammed V – Agdal, Rabat, Morocco , g , , 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (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 g es sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France h i i i f lif i i d 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales d 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energie 138 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’En 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States anta Cruz Institute for Particle Physics, University of California Santa Cruz 139 Department of Physics, University of Washington, Seattle, WA, United States 139 Department of Physics, University of Washington, Seattle, WA, United States 140 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom 141 140 Department of Physics and Astronomy, University of Sheﬃel 141 Department of Physics, Shinshu University, Nagano, Japan 142 Fachbereich Physik, Universität Siegen, Siegen, Germany 143 Department of Physics, Simon Fraser University, Burnaby, BC, Cana p f y y y 144 SLAC National Accelerator Laboratory, Stanford, CA, United States y f 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Exp Sciences, Kosice, Slovak Republic 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Sciences Kosice Slovak Republic 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius Unive y f , y f , y, ; p f y , f p y f y f Sciences, Kosice, Slovak Republic 146 (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 Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic 146 (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 Sciences, osice, Slo ak epublic 146 (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 , , p 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) Wi d J h b S h Af i 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, 146 (a) Department of Physics, University of Cape To 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Witwatersrand, Johannesburg, South Africa cs, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 147 (a) Department of Physics, Stockholm University; (b) The Oskar Klei 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, United States 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, Un 49 Departments of Physics & Astronomy and Chemistry, Stony Brook Uni 150 Department of Physics and Astronomy, University of Sussex, Brig 151 School of Physics, University of Sydney, Sydney, Australia 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 153 Department of Physics, Technion: Israel Institute of Technology, Hai p f y f gy f 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 54 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Av 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 55 Department of Physics, Aristotle University of Thessaloniki, Thessalon 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece p f y , y f , , 156 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan p f y y f 156 International Center for Elementary Particle Physics and Department of Physics, The Univers 56 International Center for Elementary Particle Physics and Department 157 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 1 8 157 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 158 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 159 Department of Physics, University of Toronto, Toronto, ON, Canada p f y y f 160 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronomy, York University, Toronto, ON, Canada 160 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronomy, York University, Toronto, ON, Canada 160 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronom p f y y 161 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan p f y y 161 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 162 Department of Physics and Astronomy, Tufts University, Medford, MA, United States 162 Department of Physics and Astronomy, Tufts University, Medford, MA, United States 163 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 64 Department of Physics and Astronomy, University of California Irvine, 164 Department of Physics and Astronomy, University of California Irvine, Irvine 164 Department of Physics and Astronomy, University of California Irvine, Irvine, CA, United States 165 (a) (b) (c) à p f y y y f f 165 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di Chimica, Fisica e Ambiente, Università 166 165 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento d 65 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP 166 Department of Physics, University of Illinois, Urbana, IL, United States 67 Department of Physics and Astronomy, University of Uppsala, Uppsala 167 Department of Physics and Astronomy, University of Uppsala, Uppsala, Swe 167 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 168 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrón 168 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamen 168 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecul de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 69 Department of Physics, University of British Columbia, Vancouver, BC, 169 Department of Physics, University of British Columbia, Vancouver, BC, Canad 170 Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 171 170 Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 171 Department of Physics, University of Warwick, Coventry, United Kingdom 172 172 Waseda University, Tokyo, Japan 30 CERN, Geneva, Switzerland Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United States 113 112 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United Sta 112 Homer L. Dodge Department of Physics and Astronomy, Univ 113 Department of Physics, Oklahoma State University, Stillw 113 Department of Physics, Oklahoma State University, Stillwater, OK, United States 113 Department of Physics, Oklahoma State University, Stillwater, OK, United States p f y y 114 Palacký University, RCPTM, Olomouc, Czech Republic ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 216 115 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 115 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 116 LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France 117 Graduate School of Science, Osaka University, Osaka, Japan 118 Department of Physics, University of Oslo, Oslo, Norway 119 Department of Physics, Oxford University, Oxford, United Kingdom p f y f y f g 120 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 120 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 120 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università d 121 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States 121 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States 121 Department of Physics, University of Pennsylvania, Phi 122 Petersburg Nuclear Physics Institute, Gatchina, Russia g y 123 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy g y 123 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fe 123 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 / y w Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. w Also at Academia Sinica Grid Computing, Institute of Physics, A w Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. x Also at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, Fra x Also at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Did y Also at School of Physical Sciences, National Institute of Science Education and Research, Bhubaneswar, India. y Also at School of Physical Sciences, National Institute of Science Education and Resea y z Also at Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. so at Dipartimento di Fisica, Sapienza Università di Roma, Roma, It z Also at Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. aa Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. aa Also at Moscow Institute of Physics and Technology State University, Dolgo ab Also at Section de Physique, Université de Genève, Geneva, Switzerland. ab Also at Section de Physique, Université de Genève, Geneva, Switzerland. ac Also at International School for Advanced Studies (SISSA), Trieste, Italy. c Also at International School for Advanced Studies (SISSA), Trieste ac Also at International School for Advanced Studies (SISSA), Trieste, Italy. d ad Also at Department of Physics and Astronomy, University of South Carolina, Columbia, SC, United States of America. ad Also at Department of Physics and Astronomy, University of South Carolina, Columbia, SC, Unite ae Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ae Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ae Also at School of Physics and Engineering, Sun Yat-sen Univers af Also at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow af Also at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Physics, M.V. Lomonosov Moscow State University, Moscow, Russia af Also at Faculty of Physics, M.V. Lomonosov Moscow State Univ ag Also at National Research Nuclear University MEPhI, Moscow, Russia. ag Also at National Research Nuclear University MEPhI, Moscow, Russia. ah Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 f Also at Department of Physics, California State University, Fresno, CA, United States of America. f Also at Department of Physics, California State University, Fresno, CA, United States of America. g Also at Tomsk State University, Tomsk, Russia. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3 i Also at Università di Napoli Parthenope, Napoli, Italy. i Also at Università di Napoli Parthenope, Napoli, Italy. j Also at Institute of Particle Physics (IPP), Canada. k Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. k Also at Department of Physics, St. Petersburg State Polytechn s, St. Petersburg State Polytechnical University, St. Petersburg, Russ k Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. l Also at Chinese University of Hong Kong, China. y g g m Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece. m Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Gree m Also at Department of Financial and Manageme n Also at Louisiana Tech University, Ruston, LA, United States of America. n Also at Louisiana Tech University, Ruston, LA, United States of America. o Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spa o Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. p Also at Department of Physics, The University of Texas at Austin, Austin, TX, United States of America. p Also at Department of Physics, The University of Texas at Austin, Austin, TX, Uni q Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. q Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. q Also at Institute of Theoretical Physics, Ilia State University, Tb r Also at CERN, Geneva, Switzerland. s Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. s Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. t Also at Manhattan College, New York, NY, United States of Am u Also at Institute of Physics, Academia Sinica, Taipei, Taiwan. u Also at Institute of Physics, Academia Sinica, Taipei, Taiwan v Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France v Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France. 172 Waseda University, Tokyo, Japan 173 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 173 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel artment of Physics, University of Wisconsin, Madison, WI, United States Department of Physics, University of Wisconsin, Madison, WI, United Sta 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 176 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 176 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 177 Department of Physics, Yale University, New Haven, CT, United St 77 Department of Physics, Yale University, New Haven, CT, United States 178 Yerevan Physics Institute, Yerevan, Armenia 178 Yerevan Physics Institute, Yerevan, Armenia Yerevan Physics Institute, Yerevan, Armenia 179 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France y 179 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France 179 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, Franc 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. b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. c Also at Novosibirsk State University, Novosibirsk, Russia. c Also at Novosibirsk State University, Novosibirsk, Russia. d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom. d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom. e Also at TRIUMF, Vancouver, BC, Canada. 217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ah Also at Institute for Particle and Nuclear Physics, Wigner Resea ah Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, B ai Also at Department of Physics, Oxford University, Oxford, United Kingdom. ai Also at Department of Physics, Oxford University, Oxford, United Kingdom. j Also at Department of Physics, Nanjing University, Jiangsu, China ak Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. ak Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. al Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United States of America. al Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United am Also at Discipline of Physics, University of KwaZulu-Natal, Durban, South Africa p y , y , , an Also at University of Malaya Department of Physics Kuala Lumpur Malaysia ∗Deceased.
https://openalex.org/W4319429966	https://www.i-jmr.org/2023/1/e41190/PDF	English	null	Strengthening the One Health Approach in the Eastern Mediterranean Region	Interactive journal of medical research	2,023	cc-by	3,871	Corresponding Author: Corresponding Author: Mirwais Amiri, MPH, MD Global Health Development \| Eastern Mediterranean Public Health Network Shmeisani, Abdallah Ben Abbas Street Bldg No 42 Amman, 11196 Jordan Phone: 962 792985349 Email: mamiri@globalhealthdev.org Corresponding Author: Mirwais Amiri, MPH, MD Global Health Development \| Eastern Mediterranean Public Health Network Shmeisani, Abdallah Ben Abbas Street Bldg No 42 Amman, 11196 Jordan Phone: 962 792985349 Email: mamiri@globalhealthdev.org INTERACTIVE JOURNAL OF MEDICAL RESEARCH INTERACTIVE JOURNAL OF MEDICAL RESEARCH Hailat et al Abstract One Health aims to use a multidisciplinary approach to combat health threats at animal, human, and environmental health interfaces. Among its broad focus areas are issues related to food safety, the control of zoonoses, laboratory services, neglected tropical diseases, environmental health, biosafety and biosecurity, and combatting antimicrobial resistance. A roundtable session was conducted on November 18, 2021, as part of the Eastern Mediterranean Public Health Network's (EMPHNET) seventh regional conference to highlight what role Global Health Development (GHD)\|EMPHNET can play to strengthen the One Health approach. This viewpoint summarizes the findings of the roundtable discussion to highlight the experts’ viewpoints on strengthening the One Health approach, including the extent of zoonotic diseases and the dynamics of pathogens and emerging diseases; the occurrence of antimicrobial-resistant pathogens as a silent pandemic; issues surrounding the globalization of trade and food safety; the importance of integrated solutions as a new norm; issues around the institutionalization and governance toward effective operationalization of the One Health approach in the region; and how the One Health approach can be operationalized at global, regional, and local levels. The panel concluded that One Health is an integrated unifying approach that aims to sustainably balance and optimize the health of people, animals, and ecosystems, and provided recommendations to strengthen the One Health approach. It also discussed how GHD\|EMPHNET can play its role in transferring the concept of One Health from theory to practice via a solid operationalization road map guide at the Eastern Mediterranean region level. The five broad priority areas of this operational guide include (1) establishing and strengthening a governance architecture, legal framework, and policy and advocacy structure for One Health operationalization in the region; (2) fostering coordination, communication, and collaboration for One Health actions across the region and beyond; (3) building the workforce capacity for effective One Health operationalization in the region; (4) supporting regional platforms for timely, effective, and efficient data sharing and exchange on all One Health–related issues; and (5) supporting risk communication, behavior change communication, and community engagement efforts in the region. (Interact J Med Res 2023;12:e41190) doi: 10.2196/41190 Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 1 (page number not for citation purposes) Strengthening the One Health Approach in the Eastern Mediterranean Region Ekhlas Hailat1, MSc, DVM; Mirwais Amiri1, MPH, MD; Nitish Debnath2, MSci, DVM, PhD; Mahmudur Rahman1, MBBS, MPHM, PhD; Md Nurul Islam1, MVM, DVM; Zahida Fatima3, DVM, PhD; Yousef Khader4, BDS, MSci, MSPH, SCD; Mohannad Al Nsour1, MD, MSci, PhD 1Global Health Development \| Eastern Mediterranean Public Health Network, Amman, Jordan 2Development Alternatives Incorporated, Dhaka, Bangladesh 3Animal Sciences Division, Pakistan Agricultural Research Council, Islamabad, Pakistan 4Department of Public Health, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan Introduction Antimicrobial resistance (AMR) occurs when bacteria, viruses, fungi, and parasites no longer respond to medications, making infections more difficult to treat [11]. AMR is a prime example of a global public health threat, which requires urgent multisectoral action [11-13]. In fact, the WHO has declared that AMR is one of the top 10 global public health threats facing humanity, citing the misuse and overuse of antibiotics as the main drivers in the development of microbial drug resistance [13]. In addition, AMR poses a significant threat to world economies, increasing mortality and disability rates, increasing longer hospital stays, and creating a need for new drug developments. The most alarming aspect of AMR is that the reduced effectiveness of antibiotics and other antimicrobials may create a future in which major surgeries and cancer chemotherapies are considered too risky [13]. While antibiotic misuse in medicine has been addressed increasingly in recent years, abuse in the agricultural sector is massively neglected and more extensive. Indeed, the Food and Drug Administration reports revealed that more than 20 million pounds of antibiotics were sold for use in livestock farms in 2014 [14] and about 80% of medically important antibiotics are regularly fed to livestock in some countries [15]. In this regard, Global Health Development (GHD)\|Eastern Mediterranean Public Health Network (EMPHNET) strongly believes in the effective role of One Health in responses and actions at the animal-human-ecosystem interface, especially targeting emerging and endemic zoonoses, and commends the role of FAO-OIE-WHO Tripartite to create and support One Health programs. A roundtable session was conducted on November 18, 2021, as part of EMPHNET’s seventh regional conference to highlight what role GHD\|EMPHNET can play to strengthen the One Health approach. This viewpoint summarizes the findings of the roundtable discussion to highlight the drivers, integrated solutions, and success stories regarding the implementation of One Health, and highlight the role that GHD\|EMPHNET can play in transferring the concept of One Health from theory to practice. Another important topic discussed during the panel revolved around the globalization of trade and food safety. It was highlighted that globalization of trade plays an important role in disease spread and food safety, posing a challenge to the public health sector [16]. FAO promotes One Health with a focus on food safety and security, sustainable agriculture, AMR, nutrition, animal and plant health, fisheries, and livelihoods. Introduction One Health has been defined by the World Health Organization (WHO) as “an approach to designing and implementing programs, policies, legislation, and research in which multiple sectors communicate and work together to achieve better public health outcomes” [1]. The importance of One Health is ever apparent in recent decades, as rapidly increasing human populations in the 21st century have led to encroachment into new geographic areas. Marked changes in climate and land use such as deforestation and intensive farming practices have resulted in more people living in close contact with domestic and wild animals [2]. Recent reports have revealed that more than 25% of original forest cover has been lost, and 75% of terrestrial environments and 66% of marine environments were severely altered by human interventions [3]. This close contact between animals and their environments creates increasing opportunities for the spilling over of pathogens between animals and people, and the rise of new diseases (ie, emerging infectious diseases [EIDs]). Furthermore, the movement of people, animals, and animal products has grown due to advances in international travel and trade, allowing these EIDs to spread easily across borders and around the world [4]. Several infectious viruses have emerged or re-emerged from wildlife, generating serious threats to the global health and the global economy. Ebola and Marburg hemorrhagic fevers, Lassa fever, dengue fever, yellow fever, West Nile fever, Zika, and chikungunya vector-borne diseases, swine flu, Middle East respiratory syndrome (MERS), and the recent COVID-19 are additional examples of zoonoses that have spread internationally, causing significant impact and creating a need for rapid intervention from scientists and public health professionals [9]. In fact, evidence suggests that SARS, MERS, and COVID-19 must serve as a wake-up call to be better prepared when facing the coming onslaught of the pathogen [10]. For effective prevention, detection, and response to EIDs or zoonotic outbreaks, communication, coordination, and collaboration among experts from all relevant fields, including public, animal, and environmental health professionals, working closely to share data and expertise is needed [1]. The WHO, Food and Agriculture Organization of the United Nations (FAO), and the World Organisation for Animal Health, formerly the Office International des Epizooties (OIE), have led the charge in promoting multisectoral responses to these issues and other public health threats at the human-animal-ecosystem interface [1,5,6]. The panel also discussed the occurrence of antimicrobial-resistant pathogens as a silent pandemic. Hailat et al Hailat et al Examples of the effects of increasing interconnectedness and the global impact of these diseases are the HIV/AIDS, severe acute respiratory syndrome (SARS), the H5N1 strain of avian influenza, and the 2009 H1N1 influenza virus pandemics. The speed by which these diseases emerge and spread causes serious economic and developmental concerns, in addition to their effects on public health. The emergence of these diseases had been concentrated in certain “hot spot” areas, like Central Africa, South and Southeast Asia, and Latin America, where compounding factors contribute to disease spread and highlight the need for the improvement of disease detection and response capacities in these countries [8]. Introduction Ensuring a One Health approach is essential for progress to anticipate (early warning), prevent, detect, and control responses to diseases that spread between animals and humans; tackle Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 2 (page number not for citation purposes) KEYWORDS One Health; operationalization; zoonosis; antimicrobial resistance, Eastern Mediterranean region countri One Health; operationalization; zoonosis; antimicrobial resistance, Eastern Mediterranean region countries Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 1 (page number not for citation purposes) https://www.i-jmr.org/2023/1/e41190 https://www.i-jmr.org/2023/1/e41190 XSL•FO RenderX INTERACTIVE JOURNAL OF MEDICAL RESEARCH Hailat et al Roundtable Panel Discussion The panel members discussed the extent of zoonotic diseases and the dynamics of pathogens and emerging diseases. It was highlighted that nearly 75% of all new or EIDs affecting humans at the beginning of the 21st century are of zoonotic origin [4]. Of those, 71.8% are reported to have genetic origins from wildlife, indicating increasing spillover in recent years [7]. https://www.i-jmr.org/2023/1/e41190 Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 2 (page number not for citation purposes) XSL•FO RenderX INTERACTIVE JOURNAL OF MEDICAL RESEARCH Hailat et al Finally, the panel discussed how the One Health approach can be operationalized at global, regional, and local levels. It was discussed that the Tripartite Zoonoses Guide (TZG), jointly developed by the FAO, OIE, and WHO to support countries in adopting a One Health approach to address zoonotic outbreaks, provides recommendations, options, and best practices, which can be used to assist countries in achieving sustainable methods for dealing with diseases with spillover potential. The TZG provides an operational guide for countries to develop the necessary capacities for preparedness for zoonotic events and efficient flow of information among concerned parties, even in low-resource settings [23]. Fortunately, One Health was adopted by the Group of Seven countries, Group of 20 countries, and World Health assemblies to reform public health, which may facilitate their implementation globally [23]. AMR; ensure food safety; prevent environment-related human and animal health threats; and combat many other health challenges arising at human-animal-environmental interfaces. Good practices from farm to fork represent a One Health approach to food safety [17]. The panel also emphasized the importance of integrated solutions as a new norm. It was highlighted that humans, animals, and environments are ever intertwined in the current globalized landscape. Therefore, a multidisciplinary approach is necessary to address any resultant emergence of zoonotic events. One Health presents a shift in the way we think about human and animal health and offers a new direction to tackle these issues, but successful implementation of interventions requires multisectoral collaboration, communication, and coordination, as well as integration. The silo mentality of certain institutions can impede the progress toward an integrated, inclusionary response and must be adjusted for effective action plans. Recommendations and Key Action or Follow-up Areas As a result of the panel discussion and the subsequent questions raised by the participants during the questions and answers session, the most essential One Health considerations and their implications specifically pertaining to the countries across the region were identified. Recommendations to strengthen the One Health approach include institutionalization and governance of the One Health strategy, securing political commitment and influencing policy changes, developing a One Health legal framework, establishing an effective coordination mechanism and promoting multisectoral collaboration, community engagement for breaking silos, and better understanding of the interconnectedness and interdependence of human-animal-ecosystem interfaces. On the other hand, it was emphasized that epidemiological data and laboratory information should be shared across sectors to ensure effective detection of and response to health threats. Joint responses to health threats should be implemented by trained One Health workforce across sectors at the local, national, regional, and global levels. Another point discussed by the panel revolved around the importance of institutionalization and the governance toward effective operationalization of the One Health approach in the region. The FAO, OIE, WHO, and the United Nations Environment Program (UNEP) have advocated the new operational definition of One Health as recommended by their advisory panel, the One Health High Level Expert Panel (OHHLEP), on December 1, 2021 [19-21]. The new One Health definition developed by the OHHLEP states “One Health is an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals, and ecosystems” [18]. It is important to stress that institutionalization and governance of the One Health approach within the government systems is crucial to reflect the success of its implementation across the region. This will only be achieved if there is enough buy-in from the countries regarding the benefits of One Health application on the public, agricultural, and economic lives of their citizens and landscapes. Ultimately, the goal is to ensure that the One Health way of thinking is sustained in all governmental operations and entities working to prevent, prepare, detect, and respond to public health events and infectious diseases. This would ensure not only more efficient national action plans but also the prosperity of nations and meeting of sustainable developmental goals [21,22]. Roundtable Panel Discussion This can be done by reframing One Health as a way to aid the smooth implementation of plans by offering a road for the cooperation of all relevant sectors and departments when handling any public health issue. When developing action plans or response programs, all concerned parties should be contacted, ideally creating a multidisciplinary team. The One Health teams can include health care providers, public health professionals, and epidemiologists representing the human health sector, and veterinarians, veterinary epidemiologists, para-veterinarians, farmers, and agriculture field workers representing animal health, in addition to ecologists and wildlife experts representing the environmental sector. One Health teams can also include law enforcement officers, social scientists, policy makers, and community members as needed to ensure effective collaboration and representation of any matter concerning the interaction at the animal-human-environment interface [18]. Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 3 (page number not for citation purposes) https://www.i-jmr.org/2023/1/e41190 Recommendations and Key Action or Follow-up Areas As a success story on the active follow-up of the panel session, an important immediate next step was to collect and synthesize all important discussion points and develop an operational guide specifically geared toward the One Health priorities of the countries across the region. Thus, in April 2022, GHD\|EMPHNET developed a technical guide entitled “Operationalization of the One Health Approach in the Eastern Mediterranean Region” to serve as a road map for One Health operationalization at the regional level [24]. The guide took into consideration the operational definition of One Health [19-21] and the most important themes that emerged from the panel discussion. Thus, for the effective operationalization of the One Health approach across the region, the following five broad priority areas were outlined: (1) establishing and strengthening a governance architecture, legal framework, and policy and advocacy structure for One Health operationalization in the region; (2) fostering coordination, communication, and collaboration for One Health actions across the region and beyond; (3) building the workforce capacity for effective One Health operationalization in the region; (4) supporting regional platforms for timely, effective, and efficient data sharing and XSL•FO RenderX Hailat et al INTERACTIVE JOURNAL OF MEDICAL RESEARCH exchange on all One Health–related issues; and (5) supporting risk communication, behavior change communication, and community engagement efforts in the region. Each of these five broader strategic areas contains additional subcomponents to allow for the development of country-specific implementation action plans. Conclusions As promoted by the Quadripartite platform of the FAO, World Organisation for Animal Health, WHO, and UNEP, One Health is an integrated unifying approach that aims to sustainably balance and optimize the health of people, animals, and ecosystems. The health of humans, domestic and wild animals, plants, and the wider environment (including ecosystems) are closely linked and interdependent. To face new health challenges that emerge at the human-animal-environment interface, collaboration, coordination, communication, and concerted action between different sectors are needed, in addition to institutionalization and governance of the One Health approach. However, many countries lack the capacity to implement such collaboration, and international organizations, nongovernmental organizations, and private sectors can help these countries. In this context, GHD\|EMPHNET can play an effective role in promoting and transferring the concept of One Health from theory to practice through its developed technical guide for the operationalization of the One Health approach at the regional level. As a further follow-up of the panel discussion, GHD\|EMPHNET will establish a regional committee on One Health. This committee will assume two main roles or dual functions: (1) ensure effective regional communication and coordination by serving primarily as a liaison between the countries in the region and relevant global entities involved in the One Health efforts and (2) ensure effective regional collaboration and capacity development by functioning as a technical advisory, support, and oversight body across the region to facilitate the One Health Quadripartite work in the region and ensure that all stakeholders across the region are actively engaged/involved in the One Health response. 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World Organisation for Animal Health. 2021. URL: https://www.oie.int/en/tripartite-and-unep-support-ohhleps-definition-of-one-health/ [accessed 2022-02-27] 21. Tripartite and UNEP support OHHLEP's definition of "One Health": Joint Tripartite (FAO, OIE, WHO) and UNEP Statement. World Health Organization. 2021. URL: https://www.who.int/news/item/ 01-12-2021-tripartite-and-unep-support-ohhlep-s-definition-of-one-health [accessed 2022-02-19] Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 5 (page number not for citation purposes) https://www.i-jmr.org/2023/1/e41190 Abbreviations Edited by A Mavragani; submitted 18.07.22; peer-reviewed by M Raimi, MM Patwary, C Zhao, A Lai, A Elidrissi; comm 28.12.22; revised version received 17.01.23; accepted 07.02.23; published 21.03.23 ©Ekhlas Hailat, Mirwais Amiri, Nitish Debnath, Mahmudur Rahman, Md Nurul Islam, Zahida Fatima, Yousef Khader, Mohannad Al Nsour. Originally published in the Interactive Journal of Medical Research (https://www.i-jmr.org/), 21.03.2023. 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 the Interactive Journal of Medical Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.i-jmr.org/, as well as this copyright and license information must be included. Interact J Med Res 2023 \| vol. 12 \| e41190 \| p. 5 (page number not for citation purposes) https://www.i-jmr.org/2023/1/e41190 XSL•FO RenderX
https://openalex.org/W652862567	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1004930&type=printable	English	null	Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers	PLOS pathogens	2,015	cc-by	14,341	Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported for the US National Institutes of Health (grants AI078799, AI089339, HL121890, AI098484, HL126554, AI116228 and AI087452 to XGY; AI098487 and AI106468 to ML). EMG is supported by the Harvard University Center of AIDS research (HU CFAR NIH/ NIAID fund 5P30AI060354-10). PBMC sample collection was supported by the Bill and Melinda Gates Foundation (OPP 1066973), the Mark and Lisa Swartz Foundation, the Ragon Institute of MGH, MIT Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers Enrique Martin-Gayo1, Maria Jose Buzon1,2, Zhengyu Ouyang1, Taylor Hickman1, Jacqueline Cronin1, Dina Pimenova1, Bruce D. Walker1,3, Mathias Lichterfeld1,2,4, Xu G. Yu1* Enrique Martin-Gayo1, Maria Jose Buzon1,2, Zhengyu Ouyang1, Taylor Hickman1, Jacqueline Cronin1, Dina Pimenova1, Bruce D. Walker1,3, Mathias Lichterfeld1,2,4, Xu G. Yu1* 1 Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts, United States of America, 2 Infectious Disease Division, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 3 Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America, 4 Infectious Disease Division, Brigham and Women’s Hospital, Boston, Massachusetts, United States of America * xyu@partners.org Editor: Guido Silvestri, Emory University, UNITED STATES Editor: Guido Silvestri, Emory University, UNITED STATES STATES Received: January 26, 2015 Accepted: May 1, 2015 Published: June 11, 2015 Copyright: © 2015 Martin-Gayo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Martin-Gayo 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. RESEARCH ARTICLE OPEN ACCESS The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly function- al HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recog- nition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune rec- ognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Im- portantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell re- sponses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes. Citation: Martin-Gayo E, Buzon MJ, Ouyang Z, Hickman T, Cronin J, Pimenova D, et al. (2015) Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV- 1 Elite Controllers. PLoS Pathog 11(6): e1004930. doi:10.1371/journal.ppat.1004930 Citation: Martin-Gayo E, Buzon MJ, Ouyang Z, Hickman T, Cronin J, Pimenova D, et al. (2015) Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV- 1 Elite Controllers. PLoS Pathog 11(6): e1004930. doi:10.1371/journal.ppat.1004930 Cell-Intrinsic Immunity to HIV in Dendritic Cells and Harvard, and the International HIV Controller Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. and Harvard, and the International HIV Controller Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. effective antiviral immune defense in EC; however, emerging studies suggest that innate and cell-intrinsic immune activities may have critical roles for supporting and enhancing HIV-1-specific T cells. Here, we performed a detailed investigation of conventional den- dritic cells (cDC) from elite controllers and their responses to HIV-1 infection. These stud- ies indicate that cDC from EC have improved abilities to sense cytosolic HIV-1 replication products, and can more effectively mount cell-intrinsic type I interferon (IFN) secretion in response to HIV-1 infection. Notably, such increased production of type I IFN in response to viral antigen translated into enhanced abilities to prime and expand HIV-1-specific T cells. Together, these data suggest that a fine-tuned interplay between innate dendritic cell responses and adaptive HIV-1-specific CD8 T cells represents a critical component of an- tiviral immune defense in elite controllers. Competing Interests: The authors have declared that no competing interests exist. Introduction Elite controllers can maintain undetectable levels of HIV-1 viral replication in the absence of antiretroviral therapy, at least in part through the generation of highly-efficient HIV-1-specific T cell responses [1–4]. As such, these patients provide living evidence that in principle, the human immune system is capable of generating a T cell-mediated immune response that al- lows to effectively controlling HIV-1 replication. However, it is uncertain why such immune responses occur only in very few patients, and what mechanisms support the development of highly-effective T cell immune responses in such a small number of individuals, and not in the majority of alternative individuals. Dendritic cells (DCs) represent the most effective naturally- occurring antigen-presenting cells and have critical roles for inducing and maintaining anti- gen-specific T cell responses [5–10]. However, specific functional characteristics of DCs that are instrumental in generating protective HIV-1-specific T- cell responses in elite controllers are unclear, and represent an understudied area of investigation. Understanding the mecha- nisms that facilitate the induction of effective HIV-1-specific T cells by dendritic cells is of criti- cal interest for developing improved immunologic approaches for HIV-1 treatment and prevention, specifically since most HIV-1 vaccine candidates rely on dendritic cells for induc- ing HIV-1-specific immune responses. g p p Human cells have the ability to respond to viral infections by cell-intrinsic immune re- sponses that lead to secretion of type I interferons (IFN-I) and upregulation of a wide panel of IFN-stimulated genes (ISG) with antiviral effector functions [11,12]. This cell-intrinsic im- mune response is extremely effective in defending the host against a panel of different viruses [13], but early reports suggested that human cells are unable to mount such immune responses against HIV-1 [14,15]. However, recent discoveries have shown that human conventional DCs (cDC) are generally capable of generating IFN-I responses to HIV-1, but HIV-1 seems to be able to escape from such cell-intrinsic immunity in most patients [16–18]. A dominant mecha- nism that may allow HIV-1 to avoid cell-intrinsic immune responses in cDC includes the ex- pression of SAMHD1, a host protein that can block HIV-1 reverse transcription by hydrolyzing dNTPs [19–22] or inhibit HIV-1 RNA through direct degradation [23]. In the presence of experimental SAMHD1 knockdown, viral replication in host cells progresses be- yond the level of reverse transcription, and viral reverse transcripts or proteins can be sensed by host molecules that can initiate secretion of type I interferons [16,24–27]. Author Summary Elite controllers (EC), a small group of HIV-1-infected individuals that are able to control viral replication in the absence of antiretroviral therapy, provide living evidence that the human immune system is able to spontaneously control HIV-1 infection and serve as a model for inducing a functional cure of HIV-1 infection in broader patient populations. Prior studies indicated that T cell-mediated immune responses represent the backbone of 1 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Exposure to HIV-1 induces rapid maturation and sustained type I IFN secretion in conventional DCs from elite controllers To determine whether human primary cDCs from EC are capable of mounting cell-intrinsic type I IFN responses against HIV-1, we conducted ex-vivo infection experiments with PBMC from different study cohorts with a GFP-encoding VSV-G pseudotyped HIV-1 virus causing single rounds of viral infections [16]; this viral construct infects cells independently of viral cor- eceptor-mediated entry processes, but can be intracellularly sensed in a similar way as R5-trop- ic primary HIV-1 isolates [27]. At 24 and 48 hours after infection, cDCs were isolated from PBMC, and subjected to gene expression analysis. Cells from untreated, chronically HIV-1-in- fected patients (CP), HIV-1-infected patients receiving suppressive antiretroviral therapy (HAART) and HIV-1-uninfected persons were used for comparison. These experiments dem- onstrated that upon exposure to HIV-1, IFNα and IFNβ mRNA expression was rapidly and significantly upregulated at 24 hours post-infection (p.i.) in cDCs from EC; these elevated levels were sustained at 48h p. i. (Fig 1A). Exposure to control viral preparations (generated by trans- fection of the viral producer cell line with salmon sperm DNA instead of the HIV-1 plasmid) failed to induce type I IFN responses. In contrast to cDC from EC, upregulation of IFNα/β ex- pression in cDC from HIV-1 negative persons was delayed and was not detectable at statistical- ly significant levels until 48h p.i.. IFNα expression in cDCs from HAART-treated patients after HIV-1 exposure was only transiently induced at 24 hours p.i. (Fig 1A); IFNβ expression was barely affected by HIV-1 infection in this cohort. In untreated CP, baseline levels of IFNα/β in cDCs were slightly elevated in comparison to alternative patients, but did not increase substan- tially upon HIV-1 exposure, neither at 24 hours nor 48 hours p. i. We subsequently analyzed the surface expression of costimulatory molecules and activation markers on cDCs after HIV-1 infection. We observed that the rapid and sustained upregulation of type I IFN expression in cDCs from EC after HIV-1 infection was associated with a signifi- cantly increased surface expression of CD86, CD83 and CD40 at 24 hours p.i., which reached lev- els otherwise observed after stimulation of cDC with the TLR3 ligand Poly(I:C) (Fig 1B and S1 Fig). cDCs from all the alternative study cohorts also upregulated costimulatory molecules and maturation markers at 48 hours after infection, indicating that they were capable of responding to the virus, but induction of these molecules was delayed in comparison to cDC from EC (S1B Fig). Cell-Intrinsic Immunity to HIV in Dendritic Cells SAMHD1, cell-intrinsic immune responses in DCs are inhibited by the host protein TREX1, a host exonuclease that degrades HIV-1 reverse transcripts, which otherwise trigger microbial DNA sensors, leading to cell-intrinsic secretion of type I interferons [18,29]. SAMHD1, cell-intrinsic immune responses in DCs are inhibited by the host protein TREX1, a host exonuclease that degrades HIV-1 reverse transcripts, which otherwise trigger microbial DNA sensors, leading to cell-intrinsic secretion of type I interferons [18,29]. In the present study, we demonstrate that unlike chronic progressors, dendritic cells from elite controllers have the ability to effectively mount cell-intrinsic type I IFN secretion in re- sponse to HIV-1 infection, likely through an accumulation of viral reverse transcripts that serve as substrates for the cytosolic DNA sensor cGAS (cyclic guanosine monophosphate- adenosine monophosphate (GMP-AMP) synthase). Recognition of these early viral replication products in elite controllers seemed to be facilitated by an enhanced upregulation of cGAS, led to rapid and sustained secretion of type I IFNs, and was functionally relevant for supporting ef- fective HIV-1-specific CD8 T cell responses. Together, these results suggest previously unrec- ognized innate mechanisms of HIV-1 immune recognition that contribute to T cell-mediated immune control of HIV-1. Introduction In this way, re- striction of HIV-1 replication by SAMHD1 may paradoxically benefit the virus more than the host, which likely explains why SAMHD1 represents the only effective HIV-1 restriction factor that HIV-1 does not neutralize through the activity of accessory proteins [28]. In addition to Human cells have the ability to respond to viral infections by cell-intrinsic immune re- sponses that lead to secretion of type I interferons (IFN-I) and upregulation of a wide panel of IFN-stimulated genes (ISG) with antiviral effector functions [11,12]. This cell-intrinsic im- mune response is extremely effective in defending the host against a panel of different viruses [13], but early reports suggested that human cells are unable to mount such immune responses against HIV-1 [14,15]. However, recent discoveries have shown that human conventional DCs (cDC) are generally capable of generating IFN-I responses to HIV-1, but HIV-1 seems to be able to escape from such cell-intrinsic immunity in most patients [16–18]. A dominant mecha- nism that may allow HIV-1 to avoid cell-intrinsic immune responses in cDC includes the ex- pression of SAMHD1, a host protein that can block HIV-1 reverse transcription by hydrolyzing dNTPs [19–22] or inhibit HIV-1 RNA through direct degradation [23]. In the presence of experimental SAMHD1 knockdown, viral replication in host cells progresses be- yond the level of reverse transcription, and viral reverse transcripts or proteins can be sensed by host molecules that can initiate secretion of type I interferons [16,24–27]. In this way, re- striction of HIV-1 replication by SAMHD1 may paradoxically benefit the virus more than the host, which likely explains why SAMHD1 represents the only effective HIV-1 restriction factor that HIV-1 does not neutralize through the activity of accessory proteins [28]. In addition to PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 2 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Exposure to HIV-1 induces rapid maturation and sustained type I IFN secretion in conventional DCs from elite controllers Addition of exogenous IFNβ to the media culture induced the expression of costimulatory molecules in a dose-dependent manner (S1C Fig) and rescued the cDC maturation defect ob- served in CP and HAART patients (S1D Fig). In addition, we observed that the expression 3 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Cell-Intrinsic Immunity to HIV in Dendritic Cells Fig 1. Type I IFN secretion and activation in cDCs after ex-vivo exposure to HIV-1. (A): IFNα and IFNβ mRNA levels in isolated BDCA1+ cDCs from H negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) and HAART-treated HIV-1 patients (HAART) at 2 and 48 hours after exposure to HIV-1 (HIV) or to media only (Med) as negative control. Horizontal lines represent the median for each specific cohort and experimental condition. (B): Mean Fluorescence Intensity (MFI) reflecting surface expression of CD86, CD83 and CD40 in cDCs from the different study cohorts at 24 hours after infection with HIV-1 (HIV) or after exposure to poly(I:C) (PIC). MFI values are expressed as fold-changes in comparison to baseli levels. Intra-individual differences were tested for statistical significance using Wilcoxon matched-pairs signed rank tests (above each cohort), differences between cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test; * p<0.05; p<0.01; * p < 0.001; **** p< 0.0001. Horizontal lines represent the median for each specific cohort and experimental condition. (C): Heatmaps reflecting gene expression patterns of 28 interferon-stimulated genes (ISG) in cDCs from Neg (n = 6), CP (n = 6) and EC (n = 6) at 48 hours after infection with HIV-1. (D): Heatmaps reflecting correlations among gene expression intensities of all 28 ISGs in indicated study cohorts. Color-coding reflects Pearson’s correlation coefficient indicating strengths of statistical association between expression intensities of given gene pairs. doi:10 1371/journal ppat 1004930 g001 Fig 1. Type I IFN secretion and activation in cDCs after ex-vivo exposure to HIV-1. (A): IFNα and IFNβ mR i (N ) i di id l i h h i i HIV 1 i f i (CP) Eli ll (EC) d HA Fig 1. Type I IFN secretion and activation in cDCs after Fig 1. Type I IFN secretion and activation in cDCs after ex-vivo exposure to HIV-1. (A): IFNα and IFNβ mRNA levels in isolated BDCA1+ cDCs from HIV- negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) and HAART-treated HIV-1 patients (HAART) at 24 and 48 hours after exposure to HIV-1 (HIV) or to media only (Med) as negative control. Horizontal lines represent the median for each specific cohort and experimental condition. Accumulation of HIV-1 RT products in cDCs from EC To explore the underlying reasons for the ability of cDCs from EC to mount early and strong cell-intrinsic immune responses upon HIV-1 infection, we subsequently analyzed HIV-1 repli- cation steps in cDCs from the different patient cohorts. In agreement with previous reports [16], productively infected cells were hardly detected in primary cDCs during the first 48 hours after exposure to HIV-1 (Fig 2A and 2B), irrespectively of the patient cohorts. Such resistance to HIV-1 was also observed in monocyte-derived DC from HIV-1 negative persons (S2A Fig), as reported previously [16]. However, we noted significant proportions of GFP-expressing, HIV-1 positive cells in primary cDCs from HIV-negative persons at 96 hours p.i. (Fig 2B and 2C) when MDDC continued to resist productive HIV-1 infection (S2A Fig). Thus, consistent with previous studies [31–33], our data indicate that primary cDCs from HIV-1 negative sub- jects are capable of supporting HIV-1 replication, at least after prolonged periods of incuba- tion. Interestingly, cDCs from both CP and HAART-treated patients displayed substantially reduced susceptibility to productive infection (Fig 2B and 2C). Remarkably, cDCs from EC also seemed less susceptible to HIV-1 infection than cDCs from healthy individuals, but a trend towards higher proportions of infected cells (Fig 2C) and higher per-cell levels of GFP ex- pression (Fig 2D) were found in cells from these patients compared to CP and HAART individ- uals. Similar results were obtained using a CCR5-tropic GFP-encoding HIV-1 virus, although as expected, efficiency of infection was lower than with the VSV-G-pseudotyped HIV-1 virus (S2B and S2C Fig). Importantly, differential susceptibility of cDCs from each cohort to infec- tion with HIV-1 was not associated with significant differences in cell viability among the study groups (S2D Fig). Together, our findings indicate that primary cDCs can be productively infected with HIV-1 in vitro and paradoxically suggest that cDCs from EC subjects are more susceptible to de novo infection with HIV-1 than CP and HAART patients. We next analyzed early steps of viral replication in primary cDCs from our study cohorts at 48 hours p.i.. Cell-Intrinsic Immunity to HIV in Dendritic Cells from EC differ from those of other patients by their ability to mount rapid and sustained cell-in- trinsic secretion of IFNα/β in response to HIV-1. from EC differ from those of other patients by their ability to mount rapid and sustained cell-in- trinsic secretion of IFNα/β in response to HIV-1. (B): Mean Fluorescence Intensity (MFI) reflecting surface expression of CD86, CD83 and CD40 in cDCs from the different study cohorts at 24 hours after infection with HIV-1 (HIV) or after exposure to poly(I:C) (PIC). MFI values are expressed as fold-changes in comparison to baseline levels. Intra-individual differences were tested for statistical significance using Wilcoxon matched-pairs signed rank tests (above each cohort), differences between cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test; * p<0.05; p<0.01; * p < 0.001; **** p< 0.0001. Horizontal lines represent the median for each specific cohort and experimental condition. (C): Heatmaps reflecting gene expression patterns of 28 interferon-stimulated genes (ISG) in cDCs from Neg (n = 6), CP (n = 6) and EC (n = 6) at 48 hours after infection with HIV-1. (D): Heatmaps reflecting correlations among gene expression intensities of all 28 ISGs in indicated study cohorts. Color-coding reflects Pearson’s correlation coefficient indicating strengths of statistical association between expression intensities of given gene pairs. doi:10.1371/journal.ppat.1004930.g001 intensity of 28 interferon-stimulated effector genes (ISG) was upregulated in cDC at 48 hours post-infection; this occurred in all patient cohorts, but was most strongly and consistently notice- able in cDC from EC, despite some heterogeneity within this group (S1 and S2 Tables and Fig 1C) [30]. Induction of most ISGs in cDCs from ECs occurred in a highly interconnected and co- ordinated fashion, while expression of individual ISG in CP and HIV-1 negative persons were more weakly correlated, and exhibited opposing increases and decreases for certain groups of transcripts (Fig 1D). Together, these findings demonstrate a unique kinetic profile of activation, maturation and IFN secretion in cDCs from EC after exposure to HIV-1, and suggest that cDCs PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 4 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 (B): Representative flow cytometry dot plots indicating GFP expression in gated CD11c+ HLA-DR+ cDCs from Neg, CP, EC and HAART individuals after 24, 48 and 96 hours of ex-vivo infection with GFP-encoding HIV-1. Numbers in dot plots reflect the proportion of GFP- positive cells within gated cDCs. (C–D): Proportion (C) and GFP MFI (D) of GFP+ cDCs from Neg, CP, EC and HAART subjects at 96 hours after infection with HIV-1 (n = 20 tested subjects for each cohort). Horizontal lines represent the median for each specific cohort and experimental condition. Differences among cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test (* p<0.05; *** p<0.001) or using Mann Whitney U test (# p<0.05; ## p<0.01). doi:10.1371/journal.ppat.1004930.g002 doi:10.1371/journal.ppat.1004930.g002 patterns in cDCs from EC that lead to a relative accumulation of RT products in cDCs, and are associated with a more effective induction of cell-intrinsic type I IFN responses. Cell-Intrinsic Immunity to HIV in Dendritic Cells Fig 2. Susceptibility of primary cDCs to ex-vivo infection with HIV-1. (A): Flow cytometry gating strategy for defining cDCs in bulk PBMC cultures. (B): Representative flow cytometry dot plots indicating GFP expression in gated CD11c+ HLA-DR+ cDCs from Neg, CP, EC and HAART individuals after 24, 48 and 96 hours of ex-vivo infection with GFP-encoding HIV-1. Numbers in dot plots reflect the proportion of GFP- positive cells within gated cDCs. (C–D): Proportion (C) and GFP MFI (D) of GFP+ cDCs from Neg, CP, EC and HAART subjects at 96 hours after infection with HIV-1 (n = 20 tested subjects for each cohort). Horizontal lines represent the median for each specific cohort and experimental condition. Differences among cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test (* p<0.05; *** p<0.001) or using Mann Whitney U test (# p<0.05; ## p<0.01). Fig 2. Susceptibility of primary cDCs to ex-vivo infection with HIV-1. (A): Flow cytometry gating strategy f d fi i DC i b lk PBMC l (B) R i fl d l i di i GFP Fig 2. Susceptibility of primary cDCs to ex-vivo infection with HIV-1. (A): Flow Fig 2. Susceptibility of primary cDCs to ex-vivo infection with HIV-1. (A): Flow cytometry gating strategy for defining cDCs in bulk PBMC cultures. (B): Representative flow cytometry dot plots indicating GFP expression in gated CD11c+ HLA-DR+ cDCs from Neg, CP, EC and HAART individuals after 24, 48 and 96 hours of ex-vivo infection with GFP-encoding HIV-1. Numbers in dot plots reflect the proportion of GFP- positive cells within gated cDCs. (C–D): Proportion (C) and GFP MFI (D) of GFP+ cDCs from Neg, CP, EC and HAART subjects at 96 hours after infection with HIV-1 (n = 20 tested subjects for each cohort). Horizontal lines represent the median for each specific cohort and experimental condition. Differences among cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test (* p<0.05; *** p<0.001) or using Mann Whitney U test (# p<0.05; ## p<0.01). Fig 2. Susceptibility of primary cDCs to ex-vivo infect Fig 2. Susceptibility of primary cDCs to ex-vivo infection with HIV-1. (A): Flow cytometry gating strategy for defining cDCs in bulk PBMC cultures. Accumulation of HIV-1 RT products in cDCs from EC Notably, despite significantly lower levels of productive HIV-1 infection in cDCs from EC compared to HIV-1 negative individuals, cDCs from EC contained similar levels of both early and late reverse transcripts and 2-LTR circles as those from HIV-1 negative individ- uals; however, in comparison to cDCs from CP and HAART-treated patients, RT products and 2-LTR circles were significantly elevated in cDC from EC (Fig 3A). Importantly, such differ- ences in HIV-1 RT product concentrations among the different study cohorts were already evi- dent at 24h p.i. (S3 Fig). In contrast, no significant difference was found in levels of integrated HIV-1 DNA in cDCs from EC in comparison to other study cohorts (Fig 3B). These data re- sulted in significantly increased ratios of late RT transcripts and 2-LTR DNA to integrated HIV-1 DNA in EC compared to CP and HAART-treated cohorts, consistent with a dispropor- tionate accumulation of RT products in cDCs from EC relative to other study cohorts (Fig 3C). Therefore, our data suggest that elevated levels of viral RT products present in cDCs from EC might facilitate cell-intrinsic viral recognition and lead to enhanced production of type I IFNs in response to HIV-1. To test whether type I IFN responses were in fact dependent on the pres- ence of HIV-1 RT products, we infected cDCs from EC with HIV-1 in the presence or absence of antiretroviral drugs inhibiting either early or late steps of HIV-1 reverse transcription or viral DNA integration. As shown in Fig 3D, pharmacological inhibition of HIV-1 reverse tran- scription dramatically reduced the expression of type I IFNs in primary cDCs. In contrast, inhi- bition of HIV-1 integration had only a modest effect on the expression of type I IFNs that did not reach statistical significance. Overall, these findings indicate distinct viral replication 5 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 (D): Inhibition of IFNα and IFNβ mRNA expression in cDCs from EC cultured in media (Med) or infected with HIV-1 (HIV) in the presence or absence of AZT, Efavirenz (EFV) or Raltegavir (Ral). Data reflect mean and standard error from qPCR values of IFNα and IFNβ mRNA levels after normalization to β-actin endogenous expression from n = 5 experiments. Numbers above bars represent the mean percentage of inhibition induced by each drug. Differences were tested for statistical significance using a one-tailed Wilcoxon matched-pairs signed rank test, * p<0.05. Fig 3. HIV-1 replication patterns in cDCs from EC. (A): Early and late HIV-1 reverse transcripts (RT) and 2-LTR circles in cDCs from indicated study subjects at 48 hours after ex vivo infection with HIV-1. (B): Analysis of integrated HIV-1 DNA in primary cDCs from the different study cohorts. Basal levels of integrated HIV-1 DNA are shown in the left panel, right panel shows de novo HIV-1 integration in cDCs after subtraction of baseline levels of integrated HIV-1 DNA. (C): Analysis of late HIV-1 RT products and 2-LTR circles normalized to levels of de novo integrated HIV-1 DNA in cDCs at 48 hours after infection. (A– C): Differences between different cohorts were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test or using Mann Whitney U test (# p<0.05; ## p<0.01). (A,B,C). Horizontal lines represent the median for each specific cohort and experimental condition. (D): Inhibition of IFNα and IFNβ mRNA expression in cDCs from EC cultured in media (Med) or infected with HIV-1 (HIV) in the presence or absence of AZT, Efavirenz (EFV) or Raltegavir (Ral). Data reflect mean and standard error from qPCR values of IFNα and IFNβ mRNA levels after normalization to β-actin endogenous expression from n = 5 experiments. Numbers above bars represent the mean percentage of inhibition induced by each drug. Differences were tested for statistical significance using a one-tailed Wilcoxon matched-pairs signed rank test, * p<0.05. doi:10.1371/journal.ppat.1004930.g003 products [19–22] or enhancing their degradation [18,29], respectively. Basal mRNA expression levels of SAMHD1 and TREX1 were not significantly different in cDCs from the different pa- tient cohorts, although a tendency for higher expression was detected in cDCs from HIV-1-in- fected patients (Fig 4A and 4B). Weak induction of SAMHD1 and LEDGF/p75 in cDCs from EC We hypothesized that altered expression patterns of intracellular host proteins might be re- sponsible for the accumulation of HIV-1 RT products observed in cDCs from EC. To investi- gate this, we first analyzed the expression of SAMHD1 and TREX1 in cDCs after exposure to HIV-1; these host factors can affect HIV-1 RT products by reducing the synthesis of HIV-1 RT 6 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Cell-Intrinsic Immunity to HIV in Dendritic Cells Fig 3. HIV-1 replication patterns in cDCs from EC. (A): Early and late HIV-1 reverse transcripts (RT) and 2-LTR circles in cDCs from indicated study subjects at 48 hours after ex vivo infection with HIV-1. (B): Analysis of integrated HIV-1 DNA in primary cDCs from the different study cohorts. Basal levels integrated HIV-1 DNA are shown in the left panel, right panel shows de novo HIV-1 integration in cDCs after subtraction of baseline levels of integrated H DNA. (C): Analysis of late HIV-1 RT products and 2-LTR circles normalized to levels of de novo integrated HIV-1 DNA in cDCs at 48 hours after infection. C): Differences between different cohorts were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test or using Mann Whitn U test (# p<0.05; ## p<0.01). (A,B,C). Horizontal lines represent the median for each specific cohort and experimental condition. (D): Inhibition of IFNα an IFNβ mRNA expression in cDCs from EC cultured in media (Med) or infected with HIV-1 (HIV) in the presence or absence of AZT, Efavirenz (EFV) or Raltegavir (Ral). Data reflect mean and standard error from qPCR values of IFNα and IFNβ mRNA levels after normalization to β-actin endogenous expression from n = 5 experiments. Numbers above bars represent the mean percentage of inhibition induced by each drug. Differences were tested for statistical significance using a one-tailed Wilcoxon matched-pairs signed rank test, * p<0.05. Fig 3. HIV-1 replication patterns in cDCs from EC. (A): Early and late HIV-1 reverse transcripts (RT) and 2-LTR circles in cDCs from indicated study subjects at 48 hours after ex vivo infection with HIV-1. (B): Analysis of integrated HIV-1 DNA in primary cDCs from the different study cohorts. Basal levels of integrated HIV-1 DNA are shown in the left panel, right panel shows de novo HIV-1 integration in cDCs after subtraction of baseline levels of integrated HIV-1 DNA. (C): Analysis of late HIV-1 RT products and 2-LTR circles normalized to levels of de novo integrated HIV-1 DNA in cDCs at 48 hours after infection. (A– C): Differences between different cohorts were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test or using Mann Whitney U test (# p<0.05; ## p<0.01). (A,B,C). Horizontal lines represent the median for each specific cohort and experimental condition. Cell-Intrinsic Immunity to HIV in Dendritic Cells Fig 4. Expression of host restriction factors in cDCs from EC. (A) SAMHD1 mRNA expression levels in cDCs from Neg (n = 11), CP (n = 11), EC (n = 11) and HAART-treated (n = 11) subjects at 48 hours after exposure to HIV-1 (HIV) or to media only (Med). (B): mRNA levels of TREX1 in cDCs from Neg (n = 11), CP (n = 11), EC (n = 11) and HAART (n = 11) individuals at 48 hours after exposure to HIV-1 (HIV), or to media only (Med). (A–B): Horizontal lines represent the median for each specific cohort and experimental condition. (C): Efficiency of siRNA-mediated SAMHD1 protein knock-down in primary cDCs 24h after treatment with scramble (SC) or SAMHD1-specific (SAM) siRNAs. Left panel shows a representative western blot analysis of SAMHD1 and β-actin expression on cDCs, right panel represents cumulative SAMHD1 expression data after normalization to β -actin in DCs nucleofected with SC (blue; n = 4) or SAM (brown; n = 4) siRNAs. The number above the bar represents the mean inhibition (%) in SAMHD1 protein expression after treatment with specific siRNAs. (D): Relative expression of early (left panel) and late (right panel) HIV-1 reverse transcripts in cDCs after ex vivo infection in the presence or absence of siRNA-mediated SAMHD1 silencing. Statistical significance was calculated using a Wilcoxon matched-pairs signed rank test. (E-F): mRNA levels of TPNO3 (E) and LEDGF/p75 (F) in cDCs from the indicated study cohorts at 48 hours after exposure to HIV-1 (HIV), or to media only (Med). Differences within cohorts were calculated using a Wilcoxon matched-pairs signed rank test * p<0.05; p<0.01; * p<0.001. (D-E-F): Horizontal lines represent the median for each specific h t d i t l diti Fig 4. Expression of host restriction factors in cDCs from EC. (A) SAMHD1 mRNA expression levels in Fig 4. Expression of host restriction factors in cDCs from EC. (A) SAMHD1 mRNA expression levels in cDCs from Neg (n = 11), CP (n = 11), EC (n = 11) and HAART-treated (n = 11) subjects at 48 hours after exposure to HIV-1 (HIV) or to media only (Med). (B): mRNA levels of TREX1 in cDCs from Neg (n = 11), CP (n = 11), EC (n = 11) and HAART (n = 11) individuals at 48 hours after exposure to HIV-1 (HIV), or to media only (Med). PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 However, after 48 hours of exposure to HIV-1, only cDCs from HIV-1 negative study persons, HAART and CP significantly induced the transcription of SAMHD1, while cDC from ECs displayed largely similar mRNA levels of SAMHD1 as unin- fected cDCs (Fig 4A). Importantly, similar patterns of SAMHD1 protein expression were also observed when cDCs were isolated prior to exposure to HIV-1 (S4A and S4B Fig). Notably, siRNA-mediated downregulation of SAMHD1 in ex-vivo infected cDCs led to an accumulation 7 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Cell-Intrinsic Immunity to HIV in Dendritic Cells of late RT products (Fig 4C and 4D). With the exception of HAART-treated patients, expres- sion levels of TREX1 were significantly increased in cDCs from all other study cohorts after HIV-1 exposure, although this was less obvious in cDCs from EC (Fig 4B). Together, these data suggest that a weaker induction of SAMHD1, and possibly of TREX1, might facilitate HIV-1 replication and contribute to accumulation of HIV-1 RT products in cDCs from EC after ex vivo infection. Since the efficiency of HIV-1 DNA integration may also affect the described accumulation of HIV-1 RT products in cDCs from EC, we subsequently analyzed transcriptional levels of host proteins supporting HIV-1 integration, such as [34–36] TPNO3 [37,38] and LEDGF/p75 [39,40]. As shown in Fig 4E, a significant increase in the amounts of TPNO3 transcripts was observed in cDCs from all patient cohorts after exposure to HIV-1. In contrast, LEDGF/p75 mRNA expression remained essentially unchanged in cDCs from EC, as opposed to cDCs from the other three patient cohorts in which upregulation of LEDGF/p75 occurred; this sug- gests that ineffective LEDGF/p75-dependent HIV-1 DNA integration in cDCs from EC may also contribute to an accumulation of RT transcripts that cannot effectively integrate into chro- mosomal DNA (Fig 4F). Thus, these data indicate that cDCs from EC have unique expression profiles of host factors relevant for shaping and structuring the HIV-1 replication cycle, and suggest that a weak induction of the host restriction factor SAMHD1, and possibly TREX1 and LEDGF/p75, in cDCs from EC may play an important role for a relative accumulation of viral reverse transcripts in cDCs from such patients. (A–B): Horizontal lines represent the median for each specific cohort and experimental condition. (C): Efficiency of siRNA-mediated SAMHD1 protein knock-down in primary cDCs 24h after treatment with scramble (SC) or SAMHD1-specific (SAM) siRNAs. Left panel shows a representative western blot analysis of SAMHD1 and β-actin expression on cDCs, right panel represents cumulative SAMHD1 expression data after normalization to β -actin in DCs nucleofected with SC (blue; n = 4) or SAM (brown; n = 4) siRNAs. The number above the bar represents the mean inhibition (%) in SAMHD1 protein expression after treatment with specific siRNAs. (D): Relative expression of early (left panel) and late (right panel) HIV-1 reverse transcripts in cDCs after ex vivo infection in the presence or absence of siRNA-mediated SAMHD1 silencing. Statistical significance was calculated using a Wilcoxon matched-pairs signed rank test. (E-F): mRNA levels of TPNO3 (E) and LEDGF/p75 (F) in cDCs from the indicated study cohorts at 48 hours after exposure to HIV-1 (HIV), or to media only (Med). Differences within cohorts were calculated using a Wilcoxon matched-pairs signed rank test * p<0.05; p<0.01; * p<0.001. (D-E-F): Horizontal lines represent the median for each specific cohort and experimental condition. doi:10.1371/journal.ppat.1004930.g004 8 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Cell-intrinsic type I IFN secretion in primary cDCs depends on cGAS We next investigated specific sensors of microbial DNA that may be involved in enhanced cell- intrinsic type I IFN secretion in response to HIV-1 infection in primary cDCs from EC. For this purpose, expression of cytosolic DNA sensors was analyzed at 24 and 48 hours after infec- tion of cDCs from different patient cohorts with HIV-1. Interestingly, we observed that the ex- pression of cGAS, a recognized sensor for HIV-1 DNA in monocyte-derived DC [27,41,42], was more efficiently upregulated in cDCs from EC shortly after infection, but expression levels in alternative patients approached those of EC after 48 hours p.i. (Fig 5A). Interestingly, the ex- pression of STING, a downstream effector of the DNA sensor cGAS [43], was also significantly induced in cDCs from EC at 24 hours p.i. (Fig 5A). Expression of IFI16, a DNA sensor impor- tant for the cell-intrinsic recognition of HIV-1 DNA in CD4 T cells [44–46], was similarly in- duced in cDCs from all cohorts at 24 hours p. i. (Fig 5A). Interestingly, expression of STING continued to increase at 48 hours in EC, but remained largely stable in the other study patient cohorts (Fig 5A). Since STING expression is sensitive to type I interferons, its continuous in- duction in cDCs from EC may result from stronger cell-intrinsic secretion of IFNα/β described above. Importantly, induction of STING and cGAS appeared to be positively correlated with upregulation of IFNβ at 48 hours p.i. on cDCs (S5A Fig), suggesting an important role of these molecules in cytoplasmic HIV-1 immune recognition. To better define the role of cGAS for the induction of type I IFN responses in cDCs from EC, we analyzed changes of type I IFN expres- sion in HIV-1-infected primary cDCs after siRNA-mediated downregulation of cGAS expres- sion. As shown in Fig 5B, silencing of cGAS (S5B and S5C Fig) drastically impaired the induction of both IFNα and IFNβ secretion in response to HIV-1 infection. These effects were not associated with changes in cell viability (S5B Fig) or an inability to respond to other stimuli such as Poly I:C (S5D Fig) in cDCs. Overall, these data suggest that the HIV-1 DNA sensor cGAS is preferentially upregulated in cDCs from EC, and facilitates a more rapid and efficient recognition of cytoplasmic HIV-1 DNA, leading to more potent cell-intrinsic IFN secretion in these cells. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Cell-Intrinsic Immunity to HIV in Dendritic Cells Fig 5. Induction of cytosolic DNA sensors in cDCs from EC. (A): Fold change in cGAS, STING and IFI16 mRNA expression levels in indicated study cohorts at 24 (upper panels) and 48 (lower panels) hours after ex- vivo infection with HIV-1. Induction of mRNA expression in comparison to baseline levels was tested for statistical significance using Wilcoxon matched-pairs signed-rank test tests. Significant differences between distinct cohorts were calculated using a Mann Whitney test. No correction for multiple comparisons was applied (B): IFNα and IFNβ expression in primary cDCs nucleofected with scrambled (SC) or cGAS-specific siRNAs, followed by infection with HIV-1. Data from n = 4 experiments are shown. Data were normalized to results from experiments with scrambled siRNA sequences. Differences in type I IFN responses between untreated or SC- or cGAS-nucleofected cDCs were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test * p<0.05; ** p<0.01. doi:10.1371/journal.ppat.1004930.g005 Fig 5. Induction of cytosolic DNA sensors in cDCs from EC. (A): Fold change in cGAS, STING and IFI16 mRNA expression levels in indicated study cohorts at 24 (upper panels) and 48 (lower panels) hours after ex- vivo infection with HIV-1. Induction of mRNA expression in comparison to baseline levels was tested for statistical significance using Wilcoxon matched-pairs signed-rank test tests. Significant differences between distinct cohorts were calculated using a Mann Whitney test. No correction for multiple comparisons was applied (B): IFNα and IFNβ expression in primary cDCs nucleofected with scrambled (SC) or cGAS-specific siRNAs, followed by infection with HIV-1. Data from n = 4 experiments are shown. Data were normalized to results from experiments with scrambled siRNA sequences. Differences in type I IFN responses between untreated or SC- or cGAS-nucleofected cDCs were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test * p<0.05; ** p<0.01. doi:10.1371/journal.ppat.1004930.g005 Cell-intrinsic type I IFN secretion in primary cDCs depends on cGAS We next investigated specific sensors of microbial DNA that may be involved in enhanced cell- intrinsic type I IFN secretion in response to HIV-1 infection in primary cDCs from EC. For this purpose, expression of cytosolic DNA sensors was analyzed at 24 and 48 hours after infec- tion of cDCs from different patient cohorts with HIV-1. Interestingly, we observed that the ex- pression of cGAS, a recognized sensor for HIV-1 DNA in monocyte-derived DC [27,41,42], 9 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 (D): Representative flow cytometry dot plots reflecting IFN-γ secretion in an HLA-A2-SL9 (SLYNTVATL)-specific CTL cell line after 16 hours of co-culture with uninfected or HIV-1-infected cDCs from Neg, CP, EC and HAART individuals. (E): Proportion of IFNγ+ cells in the SL9 CTL cell line after exposure to HIV-1-infected cDCs from indicated study groups. Cumulative data from n = 8 experiments are shown. (B,C,E): Differences within and among study groups were tested for statistical significance using a Wilcoxon matched-pairs signed-rank test rank test or a Mann Whitney test corrected for multiple comparisons using Bonferroni method, respectively. * p<0.05; ** p<0.01. (F): Induction of allogeneic CD4 (left; n = 7) and CD8 (right; n = 7) T cell proliferation after exposure to cDC from EC cultured in the presence of media (Med) or infected with HIV-1 in the presence or absence of AZT or RAL. Statistical significance of differences was tested using a Wilcoxon matched-pairs signed rank test. Bonferroni correction was applied for multiple comparisons.* p<0.05. (E-F): Horizontal lines represent the median for each specific cohort and experimental condition. Fig 6. Antigen-presenting properties of cDCs after exposure to HIV-1. (A): Representative flow cytometry plots reflecting proliferation of CD4+ T cells after stimulation with allogeneic uninfected (Med) or HIV-1 infected (HIV) cDCs from Neg, CP, EC and HAART patients. Numbers in flow cytometry plots represent the proportion of proliferating CFSElow T cells. (B–C): Induction of allogeneic CD4 (B; n = 8) and CD8 T (C; n = 7) cell proliferation after exposure to HIV-1 infected cDC from indicated study cohorts. Horizontal lines represent the median for each specific cohort and experimental condition. (D): Representative flow cytometry dot plots reflecting IFN-γ secretion in an HLA-A2-SL9 (SLYNTVATL)-specific CTL cell line after 16 hours of co-culture with uninfected or HIV-1-infected cDCs from Neg, CP, EC and HAART individuals. (E): Proportion of IFNγ+ cells in the SL9 CTL cell line after exposure to HIV-1-infected cDCs from indicated study groups. Cumulative data from n = 8 experiments are shown. (B,C,E): Differences within and among study groups were tested for statistical significance using a Wilcoxon matched-pairs signed-rank test rank test or a Mann Whitney test corrected for multiple comparisons using Bonferroni method, respectively. * p<0.05; ** p<0.01. Cell-Intrinsic Immunity to HIV in Dendritic Cells Fig 6. Antigen-presenting properties of cDCs after exposure to HIV-1. (A): Representative flow cytometry plots reflecting proliferation of CD4+ T cells after stimulation with allogeneic uninfected (Med) or HIV-1 infected (HIV) cDCs from Neg, CP, EC and HAART patients. Numbers in flow cytometry plots represent the proportion of proliferating CFSElow T cells. (B–C): Induction of allogeneic CD4 (B; n = 8) and CD8 T (C; n = 7) cell proliferation after exposure to HIV-1 infected cDC from indicated study cohorts. Horizontal lines represent the median for each specific cohort and experimental condition. (D): Representative flow cytometry dot plots reflecting IFN-γ secretion in an HLA-A2-SL9 (SLYNTVATL)-specific CTL cell line after 16 hours of co-culture with uninfected or HIV-1-infected cDCs from Neg, CP, EC and HAART individuals. (E): Proportion of IFNγ+ cells in the SL9 CTL cell line after exposure to HIV-1-infected cDCs from indicated study groups. Cumulative data from n = 8 experiments are shown. (B,C,E): Differences within and among study groups were tested for statistical significance using a Wilcoxon matched-pairs signed-rank test rank test or a Mann Whitney test corrected for multiple comparisons using Bonferroni method, respectively. * p<0.05; ** p<0.01. (F): Induction of allogeneic CD4 (left; n = 7) and CD8 (right; n = 7) T cell proliferation after exposure to cDC from EC cultured in the presence of media (Med) or infected with HIV-1 in the presence or absence of AZT or RAL. Statistical significance of differences was tested using a Wilcoxon matched-pairs signed rank test. Bonferroni correction was applied for multiple comparisons.* p<0.05. (E-F): Horizontal lines represent the median for each specific cohort and experimental condition. Fig 6. Antigen-presenting properties of cDCs after exposure to HIV-1. (A): Representative flow Fig 6. Antigen-presenting properties of cDCs after exposure to HIV-1. (A): Representative flow cytometry plots reflecting proliferation of CD4+ T cells after stimulation with allogeneic uninfected (Med) or HIV-1 infected (HIV) cDCs from Neg, CP, EC and HAART patients. Numbers in flow cytometry plots represent the proportion of proliferating CFSElow T cells. (B–C): Induction of allogeneic CD4 (B; n = 8) and CD8 T (C; n = 7) cell proliferation after exposure to HIV-1 infected cDC from indicated study cohorts. Horizontal lines represent the median for each specific cohort and experimental condition. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 doi:10.1371/journal.ppat.1004930.g006 Cell-intrinsic immune responses in cDCs from EC support HIV-1-specific T cells We sought to determine whether more efficient cell-intrinsic immune responses against HIV-1 in cDCs from EC can result in improved abilities to stimulate antigen-specific T cells. For this purpose, we first tested the ability of uninfected and HIV-1-infected cDCs from the different study cohorts to induce proliferation of CFSE-labeled allogeneic CD4+ and CD8+ T cells. As shown in Fig 6A, 6B and 6C, HIV-1-infected cDCs from EC had significantly elevated abilities to induce proliferation of allogeneic CD4+ and CD8+ T cells after 6 days in culture, compared PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 10 / 22 Discussion Cell-intrinsic secretion of type I IFN represents an efficient antimicrobial immune defense strategy, but the clinical significance of these types of immune responses against HIV-1 re- mains uncertain. Recent studies show that in principle, human cells have the ability to respond to HIV-1 infection with increased secretion of type I interferons; however, several negative reg- ulators of IFN secretion seem to actively prevent these immune responses in most patients [48–50]. In this study, we have conducted a detailed analysis of cell-intrinsic immune responses to HIV-1 in primary dendritic cells from elite controllers, a small subgroup of patients who control HIV-1 replication in the absence of treatment and arguably represent the best patients for detecting effective immune defense mechanisms against HIV-1 disease progression. Our data indicate that cDCs from EC differed from those of other patients by cell-intrinsic type I IFN secretion that occurred rapidly and sustainably after HIV-1 infection, was associated with increased cell activation, and resulted in improved abilities to stimulate HIV-1-specific CD8 T cells. As such, our data demonstrate that type I IFN responses to HIV-1 in cDCs may play an important role in the network of immune defense mechanisms in elite controllers, and delin- eate specific connections between innate HIV-1 immune recognition and the generation of adaptive immune responses in this specific patient population. Dendritic cells represent a relatively hostile environment for HIV-1 and do not effectively support HIV-1 replication steps, likely due to specific myeloid host proteins that effectively block HIV-1 replication steps. SAMHD1 is one of the most prominent myeloid restriction fac- tors, and can effectively inhibit HIV-1 reverse transcription by hydrolyzing dNTPs, which are typically only available in limited amounts in resting myeloid cells. Despite evidence that SAMHD1 sequences have been under positive selection pressure [51], it is interesting that HIV-1 has not developed effective measures to counteract the antiviral activity of this host fac- tor, suggesting that this protein may not significantly impair the ability of HIV-1 to establish a long-lasting, progressive infection of the host. Our study demonstrates the paradoxical finding that cDCs from EC are more permissive to early steps of HIV-1 replication while cDCs from patients with progressive infection are more resistant to HIV-1 infection. This corresponded to our observation that upregulation of SAMHD1 in response to HIV-1 was lower in EC, leading to more efficient reverse transcription in cDCs from these patients. Cell-Intrinsic Immunity to HIV in Dendritic Cells to HIV-1-infected cDCs from alternative study subjects. To confirm these findings with CD8+ T cells specific for HIV-1, we cultured a cytotoxic T cell (CTL) clone that recognizes the immu- nodominant HIV-1 Gag peptide SLYNTVATL restricted by HLA02:01 [47] in the presence of unstimulated or HIV-1-infected cDCs from HLA-matched study subjects. As shown in Fig 6D and 6E, HIV-1-infected cDCs from EC were able to elicit significantly more IFNγ secretion in HIV-1-specific CD8+ T cells, in contrast to cDCs from chronic progressors or HAART-treated individuals which promoted significantly less efficient IFNγ secretion. Therefore, our results indicate that upon HIV-1 infection, cDCs from EC acquire improved antigen-presenting prop- erties and have increased abilities to stimulate HIV-1-specific T cells. Importantly, improved antigen-presenting properties of cDCs from EC after infection with HIV-1 were abrogated by pharmacological inhibition of HIV-1 reverse transcription, but not by inhibition of HIV-1 inte- gration, consistent with our previous identification of viral reserve transcripts as the main viral substrate required for inducing cell-intrinsic immune responses (Fig 6F). Together, these data suggest an important contribution of innate, cell-intrinsic immune recognition of HIV-1 in cDCs to the generation of potent antiviral T cell immune responses in elite controllers. (F): Induction of allogeneic CD4 (left; n = 7) and CD8 (right; n = 7) T cell proliferation after exposure to cDC from EC cultured in the presence of media (Med) or infected with HIV-1 in the presence or absence of AZT or RAL. Statistical significance of differences was tested using a Wilcoxon matched-pairs signed rank test. Bonferroni correction was applied for multiple comparisons.* p<0.05. (E-F): Horizontal lines represent the median for each specific cohort and experimental condition. doi:10.1371/journal.ppat.1004930.g006 11 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Together these data suggest that cDCs from EC preferentially support a form of abortive HIV-1 infection that progresses effectively through reverse transcription, but is restricted at the level of viral integration, possi- bly through as of yet undefined host factors. Notably, prior studies found evidence for restric- tion of HIV-1 integration in resting CD4+ T cells from EC [54], although the underlying reason remained uncertain. The weak induction of host factors required for HIV-1 integration in cDCs from EC, such as LEDGF/p75, may represent one important aspect contributing to the accumulation of RT products in cDCs from EC, but a closer analysis of molecular mechanisms regulating and possibly restricting HIV-1 integration in cDCs from EC will likely be highly in- formative. In addition, it is interesting that higher levels of integrated HIV-1 DNA in CP and HAART-treated patients did not translate in higher levels of HIV-1 mRNA transcription, as determined by GFP expression; this suggests that additional restriction at the level of viral gene transcription may be operational in these patients. An important aspect of this work is the identification of cGAS as a critical factor for the gen- eration of cell-intrinsic type I IFN responses in primary cDCs. cGAS can act as a cytosolic mi- crobial DNA sensor that induces interferon secretion by production of the second messenger cGAMP [43,55,56] and can play an important role for innate recognition of HIV-1 DNA in myeloid cells [42]. However, in prior studies, cGAS-dependent immune recognition of HIV-1 was mostly analyzed in monocyte-derived DCs, which are highly resistant to HIV-1 unless co- transfected with viral inhibitors of SAMHD1 [27]. Our experiments in more physiologic pri- mary cDCs from EC demonstrated that HIV-1 infection led to a rapid induction of cGAS ex- pression, and cGAS silencing in primary cDCs effectively inhibited type I IFN secretion. As such, our work strongly suggests that cGAS can sense HIV-1 DNA under physiologically rele- vant conditions, and that a more rapid induction of cGAS expression facilitates the generation of cell-intrinsic type I IFN secretion in cDCs from EC. Notably, STING, an adaptor molecule that acts downstream of cGAS-dependent immune recognition [43,57], also tended to be more effectively induced in cDCs from EC, suggesting combined activity of the cGAS/STING path- way for HIV-1 immune recognition in these patients. Cell-Intrinsic Immunity to HIV in Dendritic Cells uncertain at present and requires further investigation. Overall, these data suggest that the SAMHD1-dependent restriction of HIV-1 replication in cDCs benefits the virus more than the host, and that a higher susceptibility of cDCs to HIV-1 paradoxically allows for more effective systemic immune defense against HIV-1. Notably, ISG expression and levels of IFNα/β expres- sion prior to ex-vivo infection with HIV-1 tended to be elevated in cDC from CP, consistent with higher levels of immune activation in this cohort; it is possible that this increased baseline immune activation may make cDC from these patients refractory to HIV-1-induced cell-in- trinsic immune responses. Cell-intrinsic immune responses in cDCs from EC in our study appeared to be facilitated by an accumulation of reverse transcripts. These viral products seem to represent the main viral substrate that is responsible for the induction of cellular type I IFN responses, as pharmacologi- cal inhibition of viral reverse transcripts almost completely inhibited cell-intrinsic IFN-I secre- tion, while inhibition of HIV-1 integration did not. These observations support previous data suggesting that single-stranded HIV-1 DNA transcripts represent the predominant HIV-1 rep- lication product that cell-intrinsic microbial sensors recognize [27,53]. The exact reasons re- sponsible for the accumulation of reverse transcripts in EC are not entirely clear; however, Cell-intrinsic immune responses in cDCs from EC in our study appeared to be facilitated by an accumulation of reverse transcripts. These viral products seem to represent the main viral substrate that is responsible for the induction of cellular type I IFN responses, as pharmacologi- cal inhibition of viral reverse transcripts almost completely inhibited cell-intrinsic IFN-I secre- tion, while inhibition of HIV-1 integration did not. These observations support previous data suggesting that single-stranded HIV-1 DNA transcripts represent the predominant HIV-1 rep- lication product that cell-intrinsic microbial sensors recognize [27,53]. The exact reasons re- sponsible for the accumulation of reverse transcripts in EC are not entirely clear; however, relative to the levels of reverse transcripts, HIV-1 integration in cDCs from EC was dispropor- tionately reduced, and the ratio of reverse transcripts to integrated HIV-1 DNA was exceeding- ly higher in cDCs from EC compared to all other patient cohorts. Discussion Whether this reduced in- duction of SAMHD1 in cDC from EC represents a constitutive, cell-intrinsic aspect of cDC from these patients, or occurs as a result of specific interactions with other cell types [52] is 12 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Cell-Intrinsic Immunity to HIV in Dendritic Cells While the role of highly-functional HIV-1-specific CD8 T cells for HIV-1 immune defense in elite controllers is supported by a large number of studies [58–61], the ontogeny of these ef- fective immune responses still remains largely obscure. Our data suggest that more efficient cell-intrinsic responses against HIV-1 in cDCs from EC translate into an enhanced ability to stimulate HIV-1-specific T cell responses. This may correspond to prior description to specific expression patterns of immunoregulatory receptors on cDC from EC [62]. As such, more effec- tive cell-intrinsic IFN secretion in cDCs may represent a distinguishing feature of elite control- lers, and a key mechanism supporting the evolution of effective adaptive cellular immune responses. Yet, growing evidence suggests that the ability of elite controllers to maintain unde- tectable levels of viral replication is associated with increased levels of immune activation [63,64], which may put patients at risk for higher frequencies of cardiovascular events and ac- celerated immune aging. Therefore, it is tempting to speculate that improved abilities for cyto- solic microbial immune recognition may represent a constitutive characteristic of ECs that predisposes them for more potent immune activity against HIV-1, at the expense of elevated generalized levels of immune activation. An improved understanding of interactions between innate immune recognition in cDCs, immune activation and the evolution of adaptive CD8 T cell responses in elite controllers will therefore be necessary for designing clinical strategies aiming at inducing an elite controller-like phenotype in broader populations of HIV-1 patients. Materials and Methods Study participants HIV-1 elite controllers who had maintained undetectable levels of HIV-1 replication for a me- dian of 5 years (range 2–14) in the absence of antiretroviral therapy (EC, n = 26, median VL: <49 copies/ml, median CD4 T cell counts: 835 cells/μl, range 444–2459 cells/μl), untreated chronic progressors (CP, n = 26, median VL: 9010 copies/ml, range 1360–111000; median CD4 T cell counts: 471 cells/μl, range 149–1098 cells/μl), HAART-treated chronically HIV- 1-infected patients with suppressed HIV-1 viremia (HAART, n = 23, median VL: <49 copies/ ml; median CD4 T cell counts: 710 cells/μl, range 109–1325 cells/μl) and HIV-1 seronegative healthy persons (Neg, n = 26), were recruited for this study. Ethics statement All subjects gave written informed consent and the study was approved by the Institutional Re- view Board of Massachusetts General Hospital/Partners Healthcare. Interestingly, IFI16, which can act as a cellular sensor for HIV-1 DNA in lymphoid CD4+ T cells and induces pyroptosis after abortive infection with HIV-1 [45], was similarly induced in cDCs from all cohorts early after infection, and therefore unlikely to be responsible for the early type I IFN responses observed in EC. 13 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Flow cytometry At 24, 48 and 96 hours post-infection, PBMC were stained with LIVE/DEAD cell blue viability dye (Invitrogen, Carlsbad, CA) and monoclonal antibodies directed against CD11c (Biole- gend), CD14 (BD), CD40, CD83, CD86, HLA-DR (Biolegend), and BDCA-1 (Miltenyi Biotec) and subsequently analyzed on a Fortessa cytometer (BD Biosciences, San Jose, CA). For intra- cellular cytokine staining, cells were treated with a commercial fixation/permeabilization kit (BioLegend) according to the manufacturer’s protocol. Data were analyzed with FlowJo soft- ware (Tree Star). cDCs were identified from bulk PBMCs as a population of viable CD14- lym- phocytes expressing high levels of CD11c and HLA-DR and the cDC specific marker BDCA-1. Isolation and purification of DC BDCA1+ cDCs were purified from total PBMC suspensions by MACS using anti-human BDCA-1 Kit and MS and LD columns (Miltenyi Biotec) (purity > 90%) for subsequent analysis. Cell-Intrinsic Immunity to HIV in Dendritic Cells Gene expression analysis cDNA was synthesized from total RNA obtained from purified BDCA1+ cDCs using the mir- Vana Isolation Kit (Life Technologies). Subsequently, expression of selected gene transcripts (IFNα/β, SAMHD1, TREX1, TPNO3 and LEDGF/p75) was analyzed by semiquantitative PCR using the Taqman gene expression assay (Life Technologies) with standardized primers/ probes, and normalized to the expression of the housekeeping gene ACTB (encoding β-actin). In addition, expression of 28 interferon-stimulated genes (ISG) and 3 intracellular DNA sen- sors (cGAS, STING, IFI16) was analyzed using customized 96-well plate TaqMan gene expres- sion assays (Life Technologies) using a ViiA 7 instrument (Life Technologies). Ex-vivo infection assays and cell culture PBMC or isolated cDC, without prior in vitro culture with activating agents, were infected with GFP-encoding VSV-G peudotyped HIV-1 virus (MOI = 2.4) or R5-tropic HIV-1 (Ba-L, MOI = 0.4) for 2 or 4 hours, respectively, at 37°C in the presence of 5μg/ml Polybrene (Sigma). Cells cultured with media only or with 2μg/ml TLR3 ligand Poly (I:C) were used as negative or positive control, respectively. Where indicated, PBMCs were cultured in the presence of 30ng/ ml IFNβ (PeproTech). After two washes, cells were plated at 5×105 cells per well in a 24-well plate. Where indicated, the infection assays were performed in the presence of 50nM AZT or 100nM Efavirenz or 30μM Raltegavir to specifically block generation of either early or late HIV-1 RT-products or integration, respectively. All antiretroviral agents were obtained from the NIH AIDS Reagent Program (https://www.aidsreagent.org/program_info.cfm). In addi- tion, monocyte derived dendritic cells were generated as previously described [62,67] and ana- lyzed similarly after infection with HIV-1 for control purposes. Viruses and constructs GFP-encoding R5-tropic Ba-L HIV-1 viruses and GFP-encoding Δenv NL4-3 HIV-1 viruses pseudotyped with vesicular stomatitis virus G envelope protein (VSV-G) [27,65,66], were kindly provided by Dr. Dan Littman (New York University, New York, New York, USA). Viral particles were produced by transfecting 293T cells with the respective HIV-1 plasmids and, if applicable, with pCG-VSV-G, using TransIT-293 (Mirus) in OptiMEM per the manu- facturer’s instructions. For control purposes, 293T cells were also transfected with Salmon Sperm DNA solution (Invitrogen). Supernatants were harvested 48 hours after transfection, centrifuged, filtered and treated with DNase I (20 U/ml) at room temperature for 1 hour, and stored at −80°C. 14 / 22 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Mixed leukocyte reaction assays PBMC were cultured in the presence of media or VSV-G-pseudotyped HIV-1 in the absence or presence of described concentrations of AZT or EFV or Raltegavir for 24 hours. Afterwards, BDCA1+ cDCs were purified by immunomagnetic enrichment and mixed with allogeneic total peripheral blood T lymphocytes previously stained with 5μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) at a T:DC ratio of 4:1. As a control, T cells were also cultured in the presence of media only or 2.5μg/ml PHA and 50IU/ml IL-2. After incubation for 6 days, cells were washed, stained with viability dye and anti-CD4 and anti-CD8 antibodies (Biolegend, San Diego, CA), and CFSE dilution on CD4 and CD8 T cell subpopulations was analyzed by flow cytometry using a Fortessa flow cytometer. Western blot Isolated cDCs from indicated study cohorts were lysed in RIPA buffer (Thermo Scientific) sup- plemented with Halt Protease and Phosphatase inhibitors (Thermo Scientific). Lysates were normalized for protein concentration using a Bradford assay (Bio-Rad), separated by SDS-PAGE in 4–12% Tris-Glycine gels (Novex, Life Technologies) and transferred to a PVDF membrane using the iBlot Gel Transfer system (Novex, Life Technologies). Membranes were then blocked for 30min at room temperature using Odyssey Blocking buffer (LI-COR Biosci- ences) and incubated with 1:1000 dilutions of anti-mouse β-Actin (Abcam) and either mouse anti-SAMHD1 (Clone 1F6, Origene) or rabbit anti-cGAS antibodies (clone MB21D1, Sigma), followed by secondary hybridization with goat anti-mouse (clone IRDye 800cW) and goat anti-rabbit (clone IRDye 680RD) antibodies (Odyssey). The blots were visualized using an Od- yssey imaging system (LI-COR) and the bands were quantified and normalized to β-Actin lev- els using Image Studio 3.1 software (LI-COR). siRNA-mediated gene knockdown Knockdown of the DNA sensor cGAS and the restriction factor SAMHD1 were performed by nucleofection of primary cDC (program FF137, Amaxa 4D-Nucleofector, Lonza) with specific or scramble siRNAs (Thermo scientific) according to the manufacturer’s instructions. Nucleo- fected cDC were infected with VSV-G-pseudotyped HIV-1 after 16 hours, type I IFN responses and HIV-1 reverse transcripts were subsequently analyzed at 24 hours p.i. by qPCR. Efficiency of siRNA-mediated knockdown was confirmed at the mRNA and protein levels by qPCR and Western blot, respectively (Fig 4 and S5 Fig). Cell-Intrinsic Immunity to HIV in Dendritic Cells raw qPCR data were corrected to the basal levels present in cDCs from each corresponding subject. Determination of 2-LTR circle copy numbers were calculated by extrapolating qPCR values to a standard curve generated using a plasmid harboring the sequence of 2-LTR junction and the CCR5 gene (kindly provided by Dr. Mario Stevenson, University of Miami, FL) [54]. Analysis of HIV-1 replication products Early and late HIV-1 reverse transcripts and 2-LTR circles were amplified from cell lysates as previously described [68]. Integrated HIV-1 DNA was determined using nested PCR with Alu- LTR primers as previously described [54,65,69]. Copy numbers of reverse transcripts and inte- grated HIV-1 DNA were obtained after extrapolation to specific standard curves generated from HIV-1-infected 293T cells (kindly provided by Dr. Bushman, University of Pennsylva- nia). qPCR data were normalized to relative CCR5 gene copy number. To determine the amounts of de novo integrated HIV-1 DNA and 2-LTR circles in our ex vivo infection assays, PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 15 / 22 Supporting Information S1 Fig. Expression of costimulatory molecules in cDCs from EC after ex-vivo infection with HIV-1. (A): Representative flow cytometry histograms reflecting CD86 (upper panel), CD83 (middle panel) and CD40 surface expression (lower panel) of cDCs from HIV-negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) and HAART-treated HIV-1 patients (HAART) 24h after exposure to media (full grey histograms), HIV-1 (blue histograms) or Poly I:C (purple histograms). Black histograms represent back- ground levels for each marker defined by FMO controls. (B): Mean Fluorescence Intensity (MFI) values of surface expression of CD86, CD83 and CD40 on cDCs from the different study cohorts 24h (upper panels) or 48h (lower panels) after exposure to media only (Med), HIV-1 (HIV) or to the TLR3 ligand poly(I:C) (PIC). Data on plots represent raw MFI values. Differ- ences within and between study cohorts were tested for statistical significance using Wilcoxon matched-pairs signed rank test and Mann Whitney test, respectively. Horizontal lines represent the median for each specific cohort and experimental condition. Bonferroni correction was used for multiple comparisons; p<0.05; p<0.01. (C): MFI values of surface expression of CD86, CD83 and CD40 on HIV negative cDCs after 24h in culture of media (Med) or the indi- cated concentrations of IFNβ. The plots correspond to a single experiment. (D): MFI values of surface expression of CD86, CD83 and CD40 on cDCs from the different study cohorts after 24h of culture in media only (Med) or supplemented with 30ng/ml of IFNβ. Plots represent the summary of n = 3 independent experiments. Horizontal lines represent mean values. (EPS) S2 Fig. Susceptibility of MDDCs and primary cDCs to ex vivo infection with VSV-G-pseu- dotyped or R5-tropic HIV-1. (A): Flow cytometry plots showing proportions of GFP+ MDDC at 24, 48 and 96 hours after exposure to GFP-encoding VSV-G-pseudotyped HIV-1. Numbers in plots indicate the percentage of GFP+ cells. One representative experiment out of four is shown. (B–C): Proportions (B) and GFP MFI (C) of GFP+ primary cDCs from indicated study cohorts 96 hours after exposure to R5-tropic HIV-1 virus. Horizontal lines represent the medi- an for each specific cohort and experimental condition. Differences were tested for statistical significance using a Kruskal Wallis test with post-hoc Dunn’s test ( p<0.01; p<0.001) or using Mann Whitney U test (# p<0.05; ## p<0.01). Cell-Intrinsic Immunity to HIV in Dendritic Cells Statistical analysis Significance of phenotypic differences between the different patient cohorts were assessed using Mann Whitney U tests or Wilcoxon matched-pairs signed-rank test. When appropriate, statistical analysis was corrected for multiple comparisons using a Kruskal-Wallis test with post-hoc Dunn’s test or the Bonferroni correction. To investigate expression patterns of ISGs, we used unsupervised complete linkage hierarchical cluster analysis, which groups samples to- gether by the expression similarity, and gene coexpression network analysis, which identifies the co-expression relationships among genes from different patient cohorts, by Pearson’s cor- relation coefficients [70]. In vitro stimulation of HIV-1 specific T cell responses PBMC from HLA02:01+ individuals were cultured in the presence of media or VSV-G-HIV-1 for 48 hours. Subsequently, BDCA1+ cDCs were isolated as previously described and co-cul- tured with a CD8+ T cell clone specific for the HLA02:01 restricted gag peptide SL9 (SLYNT- VATL) at a T:DC ratio of 4:1. After 1 hour of initial incubation, cells were cultured for additional 16 hours in the presence of Brefeldin A (BioLegend) and Monensin (Golgi Stop; BD Biosciences). Subsequently, intracellular expression of IFNγ (BioLegend, San Diego, CA) in the CD8 T cell clone was analyzed by flow cytometry after intracellular cytokine staining. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 16 / 22 Cell-Intrinsic Immunity to HIV in Dendritic Cells Horizontal lines represent the median for each specific cohort and experimental condition. Dif- ferences were tested for statistical significance using a Kruskal Wallis test with post-hoc Dunn’s test ( p<0.05; p<0.01) or using Mann Whitney U test (# p<0.05). (EPS) Horizontal lines represent the median for each specific cohort and experimental condition. Dif- ferences were tested for statistical significance using a Kruskal Wallis test with post-hoc Dunn’s test ( p<0.05; p<0.01) or using Mann Whitney U test (# p<0.05). (EPS) S4 Fig. SAMHD1 protein levels in primary cDCs from different study cohorts. Western blot analysis of SAMHD1 (upper panel) and β-Actin (lower panel) protein levels in isolated BDCA1+ cDCs from HIV-negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) 48h after exposure to medium (Med), HIV-1 (HIV-1) or Poly I:C (P.I:C). (A) shows results from representative patients from different experiments, (B) summarizes cumulative data from n = 5 study subjects from each cohort. (EPS) S4 Fig. SAMHD1 protein levels in primary cDCs from different study cohorts. Western blot analysis of SAMHD1 (upper panel) and β-Actin (lower panel) protein levels in isolated BDCA1+ cDCs from HIV-negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) 48h after exposure to medium (Med), HIV-1 (HIV-1) or Poly I:C (P.I:C). (A) shows results from representative patients from different experiments, (B) summarizes cumulative data from n = 5 study subjects from each cohort. (EPS) S5 Fig. cGAS is required to induce type I IFN responses in primary cDCs. (A): Spearman correlations between induction of IFNβ expression and induction of cGAS (left) and STING (right) expression levels in cDCs 48 hours after exposure to HIV-1. (B): Flow cytometry analy- sis of viability on primary cDCs 24h after nucleofection with scramble- (SC) or cGAS-specific (cGAS) siRNA. Numbers of dot plots represent the percentage of viable CD11c-positive viabili- ty dye-negative DCs. (C): Efficacy of siRNA-mediated knockdown of cGAS expression in pri- mary cDC. Data indicate mRNA (left panel) and protein (right panel) expression levels of cGAS in cDCs nucleofected with scramble- (SC) or cGAS-specific siRNAs. Relative inhibition of cGAS mRNA expression after knockdown is indicated. ( p<0.05, Wilcoxon matched-pairs signed rank test). (D): IFNβ mRNA levels present on cDCs nucleofected with SC- or cGAS-spe- cific siRNAs and cultured in the presence of media (Med) or Poly I:C (PIC). Author Contributions Conceived and designed the experiments: EMG ML XGY. Performed the experiments: EMG TH JC DP. Analyzed the data: EMG ZO MJB. Contributed reagents/materials/analysis tools: BDW. Wrote the paper: EMG ML XGY. Horizontal lines represent the median for each specific cohort and experimental condition. (EPS) S1 Table. Fold change in expression of 28 ISGs in cDCs after HIV-1 infection. (DOCX) S1 Table. Fold change in expression of 28 ISGs in cDCs after HIV-1 infection. (DOCX) S2 Table. Basal levels of 28 ISGs in cDCs from EC and CP. (DOC) S2 Table. Basal levels of 28 ISGs in cDCs from EC and CP. (DOC) PLOS Pathogens \| DOI:10.1371/journal.ppat.1004930 June 11, 2015 Supporting Information (D): Proportions of cDCs contained in CD14- lymphocytes from Neg, CP, EC and HAART after 96h of infection with a VSV-G-pseu- dotyped GFP-enconding HIV-1 virus. ( ) S3 Fig. Rapid accumulation of HIV-1 RT products in cDC from EC. 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https://openalex.org/W3038732800	https://cancerandmetabolism.biomedcentral.com/track/pdf/10.1186/s40170-020-00213-w	English	null	Metformin sensitizes therapeutic agents and improves outcome in pre-clinical and clinical diffuse large B-cell lymphoma	Cancer & metabolism	2,020	cc-by	11,093	RESEARCH Open Access Metformin sensitizes therapeutic agents and improves outcome in pre-clinical and clinical diffuse large B-cell lymphoma Anil R. Singh1†, Juan J. Gu2,3†, Qunling Zhang4, Pallawi Torka2, Suchitra Sundaram2, Cory Mavis2,3 and Francisco J. Hernandez-Ilizaliturri2,3* Abstract To view a copy of this licence, visit http://creativecommons The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: Francisco.hernandez@roswellpark.org †Anil Singh and Juan J. Gu contributed equally to this work. 2Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, USA 3Department Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article which permits use, sharing, adaptation, distributi appropriate credit to the original author(s) and t changes were made. The images or other third p licence, unless indicated otherwise in a credit lin licence and your intended use is not permitted b permission directly from the copyright holder. To The Creative Commons Public Domain Dedicatio data made available in this article, unless otherw * Correspondence: Francisco.hernandez@roswellpark.org †Anil Singh and Juan J. Gu contributed equally to this work. 2Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, USA 3Department Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, USA Full list of author information is available at the end of the article * Correspondence: Francisco.hernandez@roswellpark.org Bu a o, US Full list of author information is available at the end of the article , Full list of author information is available at the end of the article Singh et al. Cancer & Metabolism (2020) 8:10 https://doi.org/10.1186/s40170-020-00213-w Singh et al. Cancer & Metabolism (2020) 8:10 https://doi.org/10.1186/s40170-020-00213-w Abstract Background: The treatment of diffuse large B-cell lymphoma (DLBCL) is limited by the development of resistance to therapy, and there is a need to develop novel therapeutic strategies for relapsed and refractory aggressive lymphoma. Metformin is an oral agent for type 2 diabetes that has been shown to decrease cancer risk and lower mortality in other types of cancer. Methods: We performed a retrospective analysis of the RPCCC database looking at patients with DLBCL treated with front-line chemotherapy. We also performed pre-clinical studies looking at the effect of metformin on cell viability, cell number, Ki67, ATP production, apoptosis, ROS production, mitochondrial membrane potential, cell cycle, effect with chemotherapeutic agents, and rituximab. Finally, we studied mouse models to see the anti-tumor effect of metformin. Results: Among diabetic patients, metformin use was associated with improved progression-free survival (PFS) and overall survival (OS) compared to diabetic patients not on metformin. Our pre-clinical studies showed metformin is itself capable of anti-tumor effects and causes cell cycle arrest in the G1 phase. Metformin induces apoptosis, ROS production, and increased mitochondrial membrane permeability. Metformin exhibited additive/synergistic effects when combined with traditional chemotherapy or rituximab in vitro. In vivo, metformin in combination with rituximab showed improved survival compared with rituximab monotherapy. Conclusions: Our retrospective analysis showed that metformin with front-line chemotherapy in diabetic patients resulted in improved PFS and OS. Our pre-clinical studies demonstrate metformin has potential to re-sensitize resistant lymphoma to the chemo-immunotherapy and allow us to develop a hypothesis as to its activity in DLBCL. Keywords: Metformin, Rituximab-chemotherapy resistance, Lymphoma, Glucose metabolism © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 Inte which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licenc changes were made. The images or other third party material in this article are included in the article's Cre licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you permission directly from the copyright holder. Introduction study from Dr. Solomon group and a prospective study from Dr. Zhao group found that metformin use prolonged the survival of DLBCL, whereas another retrospective study conducted by Dr. Cerhan group failed to demon- strate a positive clinical benefit in the same lymphoma subtype [24–26]. Several clinical trials combined metfor- min with either sirolimus or temsirolimus with R-CHOP or DA-EPOCH-R are current evaluating in NHL [27]. The need to improve upon therapeutic approaches for re- lapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients was highlighted by the results of the prospective multicenter phase III Collaborative Trial in Relapsed Ag- gressive Lymphoma (CORAL) study. DLBCL patients pre- viously treated with rituximab (R) in combination with standard doses of cyclophosphamide, doxorubicin, vincris- tine, and prednisone (R+CHOP) had only a 34% event- free survival (EFS) after R-based salvage immunochem- otherapy followed by high-dose chemotherapy and autolo- gous stem cell transplant (HDC-ASCT) [1, 2]. Scientific efforts must be focused in defining the resistance path- ways developed by lymphoma cells and integrate thera- peutic strategies to overcome them. To this end, we developed several rituximab-resistant cell lines (RRCL) and found that the acquirement of rituximab resistance leads to resistance to multiple chemotherapy agents [3, 4]. Perhaps related to the acquirement of rituximab- chemotherapy resistance observed in our RRCL, we found a de-regulation of apoptosis, cell cycle progression, and glucose metabolism [4–7]. Cancer cells alter their mito- chondrial potential (i.e., apoptotic threshold) to resist the cytotoxic effects from the host immune-surveillance cells and/or the toxic effects of therapeutic interventions. As a consequence, the cellular metabolism shifts from aerobic to anaerobic glycolysis in order to generate adenosine tri- phosphate (ATP) (Warburg effect) [8, 9]. In an attempt to maintain adequate ATP levels and meet their energy re- quirement, those cancer cells are required to maintain a higher glucose uptake rate [10, 11]. In our current work, we present the results of a retro- spective study conducted at our Institute. In it, we found that DLBCL diabetic patients who used metformin during first-line chemo-immunotherapy had a significantly im- proved progression-free survival (PFS) and overall survival (OS) compared to non-diabetic or diabetic DLBCL pa- tients that used other glucose-lowering agent(s). Our find- ings suggested a therapeutic role of metformin in DLBCL. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 Page 2 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism Introduction Subsequently, using both, in vitro and in vivo lymphoma pre-clinical models as well as primary clinical samples ob- tained through our lymphoma clinical program, we identi- fied metformin as an effective therapeutic drug against B- cell NHL. To our knowledge, this is the first report that demonstrates metformin’s anti-cancer properties in resist- ant B-cell lymphoma. Our findings support the combin- ation of metformin with other chemotherapeutic agents as relatively inexpensive and potentially effective approach to reverse the drug-resistance in B-cell lymphoma. Differences in the clinical outcomes following first-line chemo-immunotherapy in diabetic and non-diabetic DLBCL patients. Using the RPCCC tumor registry, we identified 264 DLBCL patients treated with rituximab and anthracycline- based therapy between 1997 and 2013. The cohort of pa- tients included 49 diabetic patients. Demographic, clinical, pharmacological, and pathological characteristics were re- corded (age, sex, DLBCL subtype according to the Han’s algorithm [28], stage at diagnosis, international prognostic index [IPI] risk category, treatment type, and whether or not they received radiation as consolidation to their first line of therapy). The use of metformin or other glucose lowering agents was recorded for each diabetic patient. Differences in response rate, PFS, and OS were evaluated between non-diabetic, diabetic on metformin, or diabetic on other glucose lowering agent DLBCL patients. Metformin has been widely prescribed to type II dia- betics since 1950. The exact mechanism(s) by which met- formin lowers blood glucose levels is poorly defined. However, several hypotheses had been postulated: (1) in- hibition of hepatic gluco-neogenesis and (2) reduction of insulin resistance enhancing glucose uptake in skeletal muscle [12–14]. In addition, metformin appears to exhibit anti-cancer properties. Metformin at supra-physiological (mM range) doses is an agonist of the adenosine monophosphate-activated protein kinase (AMPK) that plays a pivotal role in cellular metabolism and B-cell de- velopment. It has been demonstrated that AMPK inhibits the mammalian target of rapamycin (mTOR), partially explaining its anti-tumor effects. Another known anti- cancer effect of metformin is through the inhibition of complex 1 in the mitochondrial electron transport chain leading to ATP reduction [15–17]. Epidemiological studies have demonstrated that the use of metformin in diabetic patients is associated with a decrease in the incidence of cancer or lower cancer-related mortality [18–23]. Changes in the expression of several cell cycle proteins in RSCL or RRCL after exposure to metformin RSCL and RRCL cells were exposed to metformin (8 mM) for 48 h, and changes in key regulators of the cell cycle pathway (C-Myc, PCNA, E2F and CDK2, (Cell Sig- naling Technologies, MA)) were evaluated using specific primary and secondary monoclonal/polyclonal anti- bodies by Western blot. Effects of metformin on transcription of C-Myc and PCNA Changes in C-Myc and PCNA expression were analyzed with a quantitative real-time PCR using TaqMan primers and probes according to the manufacturer’s directions (Applied Biosystems). Briefly, after exposure to metformin (8 or 16 mM) for 48 h, total RNA was extracted and con- verted in complementary deoxyribonucleic acid (DNA) from RSCL and RRCL using the TaqMan Gene Expres- sion cell-to-CT kit (Life Technologies) on an ABI- 7500HT (Applied Bio systems). Ct values were determined using the SDS v 2.2 software (Applied Biosystems) and compared using the ΔΔCt method. Effects of metformin on apoptosis induction, radical oxygen species (ROS) production, and changes mitochondrial potential Lymphoma cells were incubated at a cell density of 0.5 × 106/mL in complete media containing DMSO or metfor- min (16 mM). After 48 h, cells were stained with Annexin V and PI in Annexin binding buffer (Thermos Fisher, Grand Island, NY). Following staining, 10,000 events were collected on a FACScan (Becton Dickinson). Data were analyzed using the FCS express software (De Novo Software, Los Angeles, CA), and differences in apoptosis induction were compared using paired t tests in the SPSS 14.0 software (SPSS, Inc.). Cell lines and primary patient samples A panel of rituximab-sensitive (RSCL) or RRCL cell lines was cultured in RPMI1640 for the experiments as previ- ously described [3, 4]. Primary patient samples from bi- opsy specimens were procured under Roswell Park Comprehensive Cancer Center Review Board protocols I42804 and I42904. Primary neoplastic B-cells were In DLBCL, there is conflictive data on the impact of the use of metformin in DLBCL outcomes. A retrospective Page 3 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism isolated from pre-treatment biopsy tissue obtained from 16 patients with previously untreated (N = 9) or relapsed/ refractory (N = 7) NHL receiving therapy at RPCCC as previously described [29]. Effects of metformin on the cell cycle of RSCL and RRCL RSCL and RRCL were exposed to metformin (8 or 16 mM) for 48 h. Cells were then washed with PBS and fixed with 70% ethanol at – 20 °C, incubated with 100 μg/ml RNase for 30 min (Sigma-Aldrich), and stained with 50 ug/ml PI. DNA content was determined by flow cytometry. Effects of metformin on the cell cycle of RSCL and RRCL RSCL and RRCL were exposed to metformin (8 or 16 mM) for 48 h. Cells were then washed with PBS and fixed with 70% ethanol at – 20 °C, incubated with 100 μg/ml RNase for 30 min (Sigma-Aldrich), and stained with 50 ug/ml PI. DNA content was determined by flow cytometry. In vitro effect of metformin on DLBCL cell viability, cell number, ATP, and Ki67 RRCL or RSCL were exposed in vitro to escalating doses of metformin for 24, 48, or 72 h. Cells were plated at a cell density of 0.5 × 106 cells/ml. Cell proliferation was determined as the change in Presto blue (ThermoFisher, CA) reduction by living cells and measured using a FluoroScan Ascent LF (Thermo Fisher Scientific, Bar- rington, IL). The half maximal inhibition concentration (IC50) of metformin was calculated using the Graph Pad Prism Software version 6.04 (graph Pad Software, La Jolla, CA). Cell number in each condition was counted by Trypan blue exclusion. Changes in ATP production were determined using the Cell Titer-Glo Luminescent Viability Assay reagent (Promega). Experiments were done in triplicates and the percentage of ATP was assessed and normalized to controls. Ki-67 was stained using a FITC labeled mousse anti-human Ki-67 for 1 h and evaluated by flow cytometry analysis. Effect of metformin on complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC) RSCL or RRCL were exposed to metformin (4, 8, or 16 mM) or DMSO for 48 h. Subsequently, 2 × 106 viable cells were labeled with 51Cr at 37 °C, 5% CO2 for 2 h. 51Cr-labeled RSCL or RRCL were then placed in 96-well plates at a cell concentration of 1 × 105 cells/well (CMC assay) or 1 × 104 cells/well (ADCC assay). Cells were then exposed to rituximab or isotype (10 μg/ml) and hu- man serum (CMC, 1:4 dilution) or PBMCs (ADCC, 40:1 effector: target ratio) for 6 h at 37 °C and 5% CO2. 51Cr release was measured as previous described [3]. RSCL and RRCL were exposed to DMSO or metfor- min (16 mM) for 48 h. Subsequently, cells were re- suspended in 0.5 ml of PBS containing 5 μmol/l of dihy- drorhodamine 123 (Invitrogen) and incubated at 37 °C for 30 min in the dark. ROS was determined by flow cy- tometry analysis. To determine changes in the mito- chondrial potential, lymphoma cell lines were exposed to metformin (16 mM) for 48 h, and 1 × 106 cells were incubated in DiOC6 (Thermofisher) at 37 °C for 30 min. The dose of DiOC6 used (20 nM) is within the ranges suggested by standard protocols. Scientists had doses ranging between 10 and 20 nM [30, 31]. We used FCCP treatment as a positive control. Cells were then washed and re-suspended in PBS and data collected and ana- lyzed via flow cytometry. Effects of metformin on rituximab activity in vivo For the in vivo experiments, 6–8-week-old severe com- promised immunodeficiency (SCID) mice were utilized. SCID mice were inoculated on day zero with 10 × 106 Raji cells through tail vein injection. After 72 h (to allow tumor engraftment), the animals were then divided into four cohorts. The first cohort (group A) was used as control and the animals did not receive any treatment. Group B consisted of animals treated with rituximab at 10 mg/kg on day +3, +7, +10, +14. Groups C were Page 4 of 13 Page 4 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism Patients included were closely matched with regard to median age, sex, DLBCL subtype, initial stage, and IPI risk category at the time of diagnosis. The histologic subtype was closely matched among groups and was evenly divided among GCB, non-GCB, and unknown. Most patients presented with advanced stage disease (III or IV). There was a difference in the percentage of patients receiving R-DA-EPOCH in the non- diabetic patients (10.2%) compared to diabetic pa- tients (0% not on metformin vs. 3.6% on metformin). There was also a difference in the rate at which radi- ation treatment was received among non-diabetic and diabetic patients on metformin (31.6% and 32.1% re- spectively) compared to diabetic patients not on met- formin (10.0%). There was also a difference in the rate at which radiation treatment was received among non-diabetic and diabetic patients on metformin (31.6% and 32.1% respectively) compared to diabetic patients not on metformin (10.0%). There was a sta- tistically significant difference between the non- diabetic patients and the diabetic patients not on metformin (P = 0.044), otherwise the differences were not significant. treated with metformin 2 μg/ml in drinking water until the experiment finished. Groups D was treated with combination treatment of both rituximab at 10 mg/kg and metformin 2 μg/ml in drinking water. The end point of the study was survival defined as the time to development of limb paralysis. Animals that reached the end point or survived after 3 months of observation were sacrificed by cervical dislocation. The experiments were repeated on three separate occasions. From December 1997 through May 2013, 264 patients with DLBCL were treated with R-CHOP/R-CHOP-like or R-DA-EPOCH at RPCCC. These patients were identified through analysis of the RPCCC lymphoma database. Their demographic, clinical, and pathological data were compared. The following variables were measured and compared: mean age, sex, DLBCL subtype, stage at diagnosis, IPI risk category, treatment type, and whether or not they received radiation Metformin use among diabetic patients associated with improved PFS and OS to cell lines, activity was seen in samples isolated from de novo or relapsed/refractory lymphoma patients. Dif- ferences in the proliferation rate between primary tumor cells and established lymphoma cell lines could explain this finding (Fig. 2e). The use of metformin in DLBCL patients treated with front-line chemo-immunotherapy resulted in improved clinical outcomes (Fig. 1a, b). Among diabetic patients on metformin vs. diabetic patients on other glucose low- ering agents, there was a statistically significant improve- ment in both PFS (90 months vs. 60 months, P = 0.036) and OS (100 months vs. 71 months, P = 0.039). There was a trend towards improved PFS (90 months vs. 83 months, P = 0.23) and OS (100 months vs. 97 months, P = 0.25) even when comparing DM patients on metfor- min to non-diabetic patients, but this difference did not reach statistical significance. Metformin induces apoptosis, increased ROS production, and lowered mitochondrial potential Metformin induces apoptosis, increased ROS production, and lowered mitochondrial potential Mitochondria are the energy factory of the cell, but also regulate apoptosis. One of the key events in apoptosis is the loss of the mitochondrial membrane potential. The loss of the membrane potential results in caspase activa- tion, the loss of mitochondrial functions essential for cell survival as well as the release of molecules that are in- volved in caspase-independent cell death [32–34]. Once there has been a loss of the mitochondrial membrane potential, the cell has committed to undergo apoptosis. We sought to identify the effect of metformin on apop- tosis, ROS production, and mitochondrial membrane potential. The exposure of RSCL and RRCL to metfor- min resulted in an increase in apoptosis, measured by the percentage of Annexin V positive cells (Fig. 3a). While apoptosis was noted in both RSCL as well as RRCL, the effect of metformin was more pronounced in RSCL. Patient characteristics A total of 264 DLBCL patients were included in the retrospective analysis. All patients had DLBCL and re- ceived either R-CHOP, rituximab in combination with dose-adjusted etoposide, prednisone, vincristine, cyclo- phosphamide, and doxorubicin (R-DA-EPOCH) or R- CHOP-like chemotherapy (Table 1). A total of 215 patients were found to be non-diabetic and 49 pa- tients were diabetic. Among diabetic patients, 21 were not on metformin, while 28 were using metformin (may or may not have been using other agents). Table 1 Descriptive statistics of diffuse large B-cell lymphoma with or without diabetes type 1/2 treated with rituximab-doxorubicin based chemotherapy at Roswell Park Comprehensive Cancer Center Table 1 Descriptive statistics of diffuse large B-cell lymphoma with or without diabetes type 1/2 treated with rituximab-doxorubicin based chemotherapy at Roswell Park Comprehensive Cancer Center All Patients Non-Diabetic Diabetic No Metformin Diabetic Metformin Number (%) 264 215 (81.4%) 21/49 (8.0%) 28/49 (10.6%) Median Age 60 58 73 63.5 Sex F/M 92/172 (34.8%/65.2%) 73/142 (34.0%/66%) 6/15 (28.6%/71.4%) 13/15 (46.4%/53.6) Subtype GCB 70 (26.5%) 55 (25.6%) 6 (28.6%) 9 (32.1%) Non-GCB 94 (35.6%) 76 (35.3%) 8 (38.1%) 10 (35.7%) UNK 100 (37.9%) 84 (39.1%) 7 (33.3%) 9 (32.1%) Stage I-II/III-IV 94/167 (36.0%/64.0%) 79/134 (37.1%/62.9%) 7/14 (33.3%/66.6%) 8/20 (28.6%/71.4%) IPI Risk Category High 19 (7.2%) 16 (7.4%) 1 (4.8%) 2 (7.1%) High-Int 58 (22.0%) 41 (19.1%) 8 (38.1%) 9 (32.1%) Low-Int 97 (36.7%) 78 (36.3%) 8 (38.1%) 11 (39.3%) Low 90 (34.1%) 80 (37.2%) 4 (19.0%) 6 (21.4%) Treatment Type R-CHOP 231 (87.5%) 185 (86.0%) 19 (90.5%) 27 (96.4%) R-CHOP-Doxil 8 (3.0%) 6 (2.8%) 2 (9.5%) 0 (0%) R-CHOP-MTX 2 (0.8%) 2 (0.9%) 0 (0%) 0 (0%) R-EPOCH 23 (8.7%) 22 (10.2%) 0 (0%) 1 (3.6%) Radiation Not treated vs. Treated 182/78 (70.0%/30.0%) 145/67 (68.4%/31.6%) 18/2 (90.0%/10.0%) 19/9 (67.9%/32.1%) From December 1997 through May 2013, 264 patients with DLBCL were treated with R-CHOP/R-CHOP-like or R-DA-EPOCH at RPCCC. These patients were identified through analysis of the RPCCC lymphoma database. Their demographic, clinical, and pathological data were compared. Patient characteristics The following variables were measured and compared: mean age, sex, DLBCL subtype, stage at diagnosis, IPI risk category, treatment type, and whether or not they received radiation Table 1 Descriptive statistics of diffuse large B-cell lymphoma with or without diabetes type 1/2 treated with based chemotherapy at Roswell Park Comprehensive Cancer Center s of diffuse large B-cell lymphoma with or without diabetes type 1/2 treated with rituximab-doxorubicin well Park Comprehensive Cancer Center Singh et al. Cancer & Metabolism (2020) 8:10 Page 5 of 13 (2020) 8:10 Singh et al. Cancer & Metabolism Fig. 1 Effects of the use metformin during first-line chemo-immunotherapy in diffuse large B-cell lymphoma patients. Retrospective analysis of 264 patients at RPCCC diagnosed with DLBCL. Kaplan-Meier curves showing an improved progression-free survival (PFS) (a) and overall survival (OS) (b) based on metformin use in diabetic DLBCL patients. When the impact of metformin use during rituximab and systemic chemotherapy (doxorubicin-based poly-chemotherapy) was compared among all patients, diabetic patients on metformin (red line) vs. other glucose lowering agents (green line) had a statistically significant improvement in both PFS (90 months vs. 60 months, P = 0.036) and OS (100 months vs. 71 months, P = 0.039). There was a trend towards improved PFS (90 months vs. 83 months, P = 0.23) and OS (100 months vs 97 months, P = 0.25) even when compared to non-diabetic patients (blue line), but this difference did not reach statistical significance Fig. 1 Effects of the use metformin during first-line chemo-immunotherapy in diffuse large B-cell lymphoma patients. Retrospective analysis of 264 patients at RPCCC diagnosed with DLBCL. Kaplan-Meier curves showing an improved progression-free survival (PFS) (a) and overall survival (OS) (b) based on metformin use in diabetic DLBCL patients. When the impact of metformin use during rituximab and systemic chemotherapy (doxorubicin-based poly-chemotherapy) was compared among all patients, diabetic patients on metformin (red line) vs. other glucose lowering agents (green line) had a statistically significant improvement in both PFS (90 months vs. 60 months, P = 0.036) and OS (100 months vs. 71 months, P = 0.039). There was a trend towards improved PFS (90 months vs. 83 months, P = 0.23) and OS (100 months vs 97 months, P = 0.25) even when compared to non-diabetic patients (blue line), but this difference did not reach statistical significance Metformin use among diabetic patients associated with improved PFS and OS Metformin single agent results in time and dose- dependent killing and decreased cellular proliferation IC50 results were obtained from GraphPad Prism6 after metformin 72 h treatment. b RSCL and RRCL cells were cultured in the presence or absence of metformin 16 mM for 48 h. Cell number in each condition was counted by Trypan blue. c Ki-67 was stained followed by flow cytometry analysis in RSCL and RRCL exposed to DMSO control or metformin (16 mM) for 48 h. d Cells were exposed to metformin 16 mM for 48 hours, and then ATP levels in each of cell lines were assessed by Titer Glo and normalized to the cell number. ATP production was calculated as percentage to the untreated control (100%). e Primary tumor cells isolated from patients with either newly diagnosed or relapsed/refractory B-cell lymphoma cells were exposed to metformin (16 mM) for 48 h. Titer Glo assay was performed to assess growth inhibition. The results are determined as percentage of viable cells compared with control. Data represented mean ± SD derived from 3 independent experiments Fig. 2 Metformin inhibited rituximab-sensitive and resistant B-cell lymphoma cell lines viability. a RSCL and RRCL were treated with accumulative concentration of metformin for 24, 48, or 72 h, respectively. Presto blue assays were performed to assess growth inhibition of metformin. The results are determined as percentage of viable cells compared with control. IC50 results were obtained from GraphPad Prism6 after metformin 72 h treatment. b RSCL and RRCL cells were cultured in the presence or absence of metformin 16 mM for 48 h. Cell number in each condition was counted by Trypan blue. c Ki-67 was stained followed by flow cytometry analysis in RSCL and RRCL exposed to DMSO control or metformin (16 mM) for 48 h. d Cells were exposed to metformin 16 mM for 48 hours, and then ATP levels in each of cell lines were assessed by Titer Glo and normalized to the cell number. ATP production was calculated as percentage to the untreated control (100%). e Primary tumor cells isolated from patients with either newly diagnosed or relapsed/refractory B-cell lymphoma cells were exposed to metformin (16 mM) for 48 h. Titer Glo assay was performed to assess growth inhibition. The results are determined as percentage of viable cells compared with control. Data represented mean ± SD derived from 3 independent experiments lines (RSCL and RRCL), metformin resulted in an in- crease in ROS production. Metformin single agent results in time and dose- dependent killing and decreased cellular proliferation Unlike the Raji and RL cell clines, the U2392 cell lines did not show an increase in ROS production when exposed to metformin. Lastly, we examined the effect of metformin on the mitochondrial membrane potential (Fig. 3c). The loss in mitochondrial membrane potential was more pronounced in RSCL al- though it was seen in both RSCL and RRCL. metformin exposure led to cell cycle arrested in the G1 phase and a decrease in the S phase (Fig. 4a). This effect was seen in all cell lines and among RSCL as well as RRCL; however, the effect was the greatest among the Raji and RL (GCB) cell lines compared to U2392 (non-GCB). To further characterize how metformin affects the cell cycle distribution in lymphoma cells, we study changes in G1 cell cycle regulatory proteins following drug exposure (Fig. 4b). In Raji and RL cells, in vitro exposure to metformin resulted in a decrease in C-Myc, PCNA, E2F, and CDK2 as detected by Western blotting. We further explored the mRNA changes of C-Myc and PCNA after exposure to metformin (Fig. 4c, d). Exposure to metformin resulted in a marked decrease in the mRNA levels of C-Myc and PCNA in the Raji and RL cell lines consistent with what was observed in the Western blots. Metformin single agent results in time and dose- dependent killing and decreased cellular proliferation The addition of metformin to RSCL and RRCL resulted in a dose and time-dependent decrease in cell viability (Fig. 2a). Metformin was also directly cytotoxic to cells, resulting in decreased cell number (Fig. 2b) and de- creased proliferative index (Ki-67 index), in both RSCL and RRCL (Fig. 2c). The exposure of metformin resulted in a reduction of ATP (Fig. 2d) in RSCL and RRCL. We explored if metformin induced cell death in primary tumor cells isolated from lymphoma patients (N = 16, Supplemental Table 1). To lesser degree when compared Subsequently, we examined the effect of metformin on the production of ROS (Fig. 3b). In both Raji and RL cell Singh et al. Cancer & Metabolism Singh et al. Cancer & Metabolism (2020) 8:10 Page 6 of 13 Fig. 2 Metformin inhibited rituximab-sensitive and resistant B-cell lymphoma cell lines viability. a RSCL and RRCL were treated with accumulative concentration of metformin for 24, 48, or 72 h, respectively. Presto blue assays were performed to assess growth inhibition of metformin. The results are determined as percentage of viable cells compared with control. IC50 results were obtained from GraphPad Prism6 after metformin 72 h treatment. b RSCL and RRCL cells were cultured in the presence or absence of metformin 16 mM for 48 h. Cell number in each condition was counted by Trypan blue. c Ki-67 was stained followed by flow cytometry analysis in RSCL and RRCL exposed to DMSO control or metformin (16 mM) for 48 h. d Cells were exposed to metformin 16 mM for 48 hours, and then ATP levels in each of cell lines were assessed by Titer Glo and normalized to the cell number. ATP production was calculated as percentage to the untreated control (100%). e Primary tumor cells isolated from patients with either newly diagnosed or relapsed/refractory B-cell lymphoma cells were exposed to metformin (16 mM) for 48 h. Titer Glo assay was performed to assess growth inhibition. The results are determined as percentage of viable cells compared with control. Data represented mean ± SD derived from 3 independent experiments Fig. 2 Metformin inhibited rituximab-sensitive and resistant B-cell lymphoma cell lines viability. a RSCL and RRCL were treated with accumulative concentration of metformin for 24, 48, or 72 h, respectively. Presto blue assays were performed to assess growth inhibition of metformin. The results are determined as percentage of viable cells compared with control. Metformin induces G1 cell cycle arrest in both RSCL and RRCL Low DiOC6 population was gated for low mitochondrial outer membrane potential (ΔΨm-Low) and FCCP was used as a positive control. All data represented mean ± SD derived from 3 independent experiments d d d ROS d d d d h h d l l RSCL d Fig. 3 Metformin induced apoptosis, increased ROS production, and decreased the mitochondrial potential in vitro. a RSCL and RRCL were exposed to control or metformin (16 mM) for 48 h, and then the percentage of cells undergoing apoptosis (Annexin V + cells) was determined by flow cytometry. Asterisk indicates that the differences were significant at P < 0.05. b Oxidative stress induced by metformin was determined by comparing dihydrorhodamine-123 (DHR-123) fluorescence intensity at 24 h post-metformin exposure to DHR-123 fluorescence of controls. All data were normalized to the control cell as 100% ROS production. Data represented mean ± SD derived from 3 independent experiments. Asterisk means P < 0.05. c RSCL and RRCL were cultured for 48 h in the absence or presence of metformin (16 mM). Subsequently, cells were stained for 30 min at 37 °C with DiOC6 followed by flow cytometry analysis. Low DiOC6 population was gated for low mitochondrial outer membrane potential (ΔΨm-Low) and FCCP was used as a positive control. All data represented mean ± SD derived from 3 independent experiments Fig. 3 Metformin induced apoptosis, increased ROS production, and decreased the mitochondrial potential in vitro. a RSCL and RRCL were exposed to control or metformin (16 mM) for 48 h, and then the percentage of cells undergoing apoptosis (Annexin V + cells) was determined by flow cytometry. Asterisk indicates that the differences were significant at P < 0.05. b Oxidative stress induced by metformin was determined by comparing dihydrorhodamine-123 (DHR-123) fluorescence intensity at 24 h post-metformin exposure to DHR-123 fluorescence of controls. All data were normalized to the control cell as 100% ROS production. Data represented mean ± SD derived from 3 independent experiments. Asterisk means P < 0.05. c RSCL and RRCL were cultured for 48 h in the absence or presence of metformin (16 mM). Subsequently, cells were stained for 30 min at 37 °C with DiOC6 followed by flow cytometry analysis. Low DiOC6 population was gated for low mitochondrial outer membrane potential (ΔΨm-Low) and FCCP was used as a positive control. All data represented mean ± SD derived from 3 independent experiments Metformin induces G1 cell cycle arrest in both RSCL and RRCL Prior studies [35–37] had shown that in vitro addition of metformin to solid tumor cancer cells resulted in cell cycle arrest. To further investigate the effect of metfor- min on the cell cycle in lymphoma cells, we performed flow cytometric analysis of cells in response to the treat- ment with metformin for 48 h. In both RSCL and RRCL, Singh et al. Cancer & Metabolism (2020) 8:10 Page 7 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Fig. 3 Metformin induced apoptosis, increased ROS production, and decreased the mitochondrial potential in vitro. a RSCL and RRCL were exposed to control or metformin (16 mM) for 48 h, and then the percentage of cells undergoing apoptosis (Annexin V + cells) was determined by flow cytometry. Asterisk indicates that the differences were significant at P < 0.05. b Oxidative stress induced by metformin was determined by comparing dihydrorhodamine-123 (DHR-123) fluorescence intensity at 24 h post-metformin exposure to DHR-123 fluorescence of controls. All data were normalized to the control cell as 100% ROS production. Data represented mean ± SD derived from 3 independent experiments. Asterisk means P < 0.05. c RSCL and RRCL were cultured for 48 h in the absence or presence of metformin (16 mM). Subsequently, cells were stained for 30 min at 37 °C with DiOC6 followed by flow cytometry analysis. Low DiOC6 population was gated for low mitochondrial outer membrane potential (ΔΨm-Low) and FCCP was used as a positive control. All data represented mean ± SD derived from 3 independent experiments Fig. 3 Metformin induced apoptosis, increased ROS production, and decreased the mitochondrial potential in vitro. a RSCL and RRCL were exposed to control or metformin (16 mM) for 48 h, and then the percentage of cells undergoing apoptosis (Annexin V + cells) was determined by flow cytometry. Asterisk indicates that the differences were significant at P < 0.05. b Oxidative stress induced by metformin was determined by comparing dihydrorhodamine-123 (DHR-123) fluorescence intensity at 24 h post-metformin exposure to DHR-123 fluorescence of controls. All data were normalized to the control cell as 100% ROS production. Data represented mean ± SD derived from 3 independent experiments. Asterisk means P < 0.05. c RSCL and RRCL were cultured for 48 h in the absence or presence of metformin (16 mM). Subsequently, cells were stained for 30 min at 37 °C with DiOC6 followed by flow cytometry analysis. Metformin enhances cytotoxic killing by chemotherapy and rituximab’s activity in vitro and in vivo We then tested to see if metformin could enhance the activity of rituximab by exposing RSCL and RRCL to varying concentrations of metformin and then evaluated complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC) in the presence of rituximab (Fig. 5c). RSCL showed increased CMC in a dose-dependent manner to metformin. There was no effect on CMC in RRCL. The addition of metfor- min with rituximab resulted in increased ADCC in both RSCL and RRCL. To examine if metformin could enhance the anti-tumor activity of chemotherapy drugs, we next tested cell via- bility with RSCL and RRCL after exposure to metformin and/or chemotherapy agents. Synergistic activity was ob- served when metformin was combined with doxorubicin (Fig. 5a) in RSCLs (the combination index (CI) of Raji is 0.7 and CI of RL is 0.8). There was also a trend to- wards improvement in the U2392 and the RRCL, but these did not reach statistical significance. The addition of metformin to dexamethasone (Fig. 5b) resulted in a statistically significant decrease in cell viability in all cell lines tested except the U2392 cell line. The CI value calculated by the CalcuSyn software was less than 0.5 (Fig. 5c), which indicated a synergistic effect be- tween metformin and dexamethasone among our RSCL and RRCL. The treatment of human lymphoma-bearing SCID mice with rituximab resulted in prolongation in survival as compared with placebo-treated controls (Fig. 5d). The administration of metformin in combination with rituxi- mab resulted in the most effective anti-tumor activity and prolongation of survival of human lymphoma- bearing SCID mice. Statistically significant differences were observed between animals treated with rituximab Page 8 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism Fig. 4 Metformin inhibited G1 cell cycle regulators and induces G1 cell cycle arrest in both RSCL and RRCL. a RSCL and RRCL were exposed to DMSO or metformin (16 mM) for 48 h, and then cell cycle distribution changes was determined by flow cytometry. Cumulative results of G1/S/ G2-M phases are shown as mean ± standard error of the mean (SEM) (n = 3). Column, results are mean + SD of 3 independent experiments. Asterisk means P < 0.05. b Effect of metformin on cell cycle proteins. Western blot of G1 cell cycle regulatory proteins levels in metformin- induced RSCL and RRCL. c, d Effects of metformin on transcriptional levels of C-Myc and PCNA. Metformin enhances cytotoxic killing by chemotherapy and rituximab’s activity in vitro and in vivo Cells were treated with metformin 16 mM for 24 h. C-Myc and PCNA mRNA levels were analyzed by real-time qPCR Fig. 4 Metformin inhibited G1 cell cycle regulators and induces G1 cell cycle arrest in both RSCL and RRCL. a RSCL and RRCL were exposed to DMSO or metformin (16 mM) for 48 h, and then cell cycle distribution changes was determined by flow cytometry. Cumulative results of G1/S/ G2-M phases are shown as mean ± standard error of the mean (SEM) (n = 3). Column, results are mean + SD of 3 independent experiments. Asterisk means P < 0.05. b Effect of metformin on cell cycle proteins. Western blot of G1 cell cycle regulatory proteins levels in metformin- induced RSCL and RRCL. c, d Effects of metformin on transcriptional levels of C-Myc and PCNA. Cells were treated with metformin 16 mM for 24 h. C-Myc and PCNA mRNA levels were analyzed by real-time qPCR chemotherapy. We identified E2F as a potential target of metformin that has not been previously identified. Based on our research, we hypothesize a model for the anti- tumor mechanism for metformin (Fig. 6). vs. metformin plus rituximab. The median survival time of animals treated with metformin and rituximab was longer (72.5 ± 5.11 days) than those treated with rituxi- mab monotherapy alone (median survival of 51.5 ± 2.6 days); log-rank test, P = 0.01. g In pre-clinical modes, we saw that metformin as a sin- gle agent was able to affect time and dose-dependent killing in RSCL as well as RRCL. This was shown in both cell lines as well as in patient-derived primary cells. The time and dose-dependent response were found at met- formin IC50 of 7–13 mM. The metformin drug concen- trations utilized in our in vitro experiments are higher than the estimated drug concentrations achieved in our in vivo experiments. These discrepancies were also noted in other scientific contributions. The higher dose of met- formin needed for in vitro experiments could be partially due to the media used during the experimental design. As described by Heiden et al., pyruvate, glucose, and as- partate which are common ingredients in culture media (i.e., DMEM and RPMI1640) can diminish metformin sensitivity [38]. Another second possible explanation to explain the higher doses of metformin required in in vitro experiments, in our rituximab-chemotherapy- Discussion RSCL and RRCL were exposed to metformin for 48 h and changes in cell viability assessed by the change in Presto blue reduction as measured by flow cytometry. Data represented mean ± SD derived from 3 independent experiments. Asterisk means P < 0.05. c Metformin enhanced anti-CD20 antibody-mediated complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC) in RSCL and RRCL. Cells were exposed to media or metformin for 24 h and subsequently labeled with 51Cr. Labeled cells were then exposed to isotype or anti-CD20 antibodies, and 20% human serum pooled from healthy volunteers (CMC) or peripheral blood mononuclear cells at an effector:target ratio of 40:1 (ADCC) and incubated at 37 °C, 5% CO2 for 6 h. 51Cr release was measured and the percentage of lysis was calculated. Asterisk means P < 0.05. d Metformin potentiated the anti-tumor activity of rituximab in mouse. Survival differences between groups were compared using log-rank analysis. Experiments were repeated in three separate times. The survival difference between rituximab as a single agent compared to rituximab combined with metformin at 2 μg/ml dosage was found to be significant (P = 0.01) resistant cell lines, is their known abnormal metabolic state and apoptotic threshold [7]. cells that were more dependent on glycolysis were in fact more susceptible to inhibition of the complex I, while there was reduced or absent effect on normal cells [41]. Our research shows that in DLBCL RSCL as well as RRCL, the addition of metformin resulted in a decrease in ATP production which may be responsible for the de- creased cellular proliferation seen in RSCL and RRCL treated with metformin. Cellular proliferation, as measured by Ki-67, has been associated with worse outcomes in DLBCL [39]. In all cell lines, the exposure to metformin resulted in a de- crease in the Ki-67. Metformin’s effects on mitochondria are complex, but one of its mechanisms is the inhibition of complex I of the electron transport chain [37, 40]. Prior studies in breast cancer models have shown that metformin causes a decrease in ATP production, which subsequently leads to a decrease in cellular proliferation [37]. Cancer cells typically shift their metabolism to an- aerobic glycolysis to help them produce ATP even in an- aerobic environments (Warburg’s effect) [17, 41]. Research in breast cancer models suggested that cancer The effect of metformin on cellular proliferation has been shown in several other malignant cell lines [42]. Discussion Our contribution highlights the effect of metformin in aggressive B-cell lymphoma starting with a retrospective analysis in DLBCL patients, and then working backwards using pre-clinical models. This study is relevant for fu- ture drug design, as well as clinical study design for pa- tients with DLBCL, particularly in underdeveloped countries where access to costly treatments are an on- going challenge. Our initial research was a retrospective analysis of a large cohort of patients with DLBCL at a single institution where we found that the use of metfor- min was associated with improved clinical outcomes. We then performed studies on pre-clinical models where we showed that metformin acts via several mechanisms to both exert anti-tumor effects independently, as well as to enhance the anti-tumor effects of traditional Page 9 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism Fig. 5 Metformin enhanced chemotherapy and rituximab’s activity in vitro and in vivo. a, b Metformin enhances doxorubicin and dexamethasone anti-tumor killing activity in RSCL and RRCL. RSCL and RRCL were exposed to metformin for 48 h and changes in cell viability assessed by the change in Presto blue reduction as measured by flow cytometry. Data represented mean ± SD derived from 3 independent experiments. Asterisk means P < 0.05. c Metformin enhanced anti-CD20 antibody-mediated complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC) in RSCL and RRCL. Cells were exposed to media or metformin for 24 h and subsequently labeled with 51Cr. Labeled cells were then exposed to isotype or anti-CD20 antibodies, and 20% human serum pooled from healthy volunteers (CMC) or peripheral blood mononuclear cells at an effector:target ratio of 40:1 (ADCC) and incubated at 37 °C, 5% CO2 for 6 h. 51Cr release was measured and the percentage of lysis was calculated. Asterisk means P < 0.05. d Metformin potentiated the anti-tumor activity of rituximab in mouse. Survival differences between groups were compared using log-rank analysis. Experiments were repeated in three separate times. The survival difference between rituximab as a single agent compared to rituximab combined with metformin at 2 μg/ml dosage was found to be significant (P = 0.01) Fig. 5 Metformin enhanced chemotherapy and rituximab’s activity in vitro and in vivo. a, b Metformin enhances doxorubicin and dexamethasone anti-tumor killing activity in RSCL and RRCL. Discussion Our findings are consistent with other investigators. The mechanisms by which metformin affects cellular prolif- eration in DLBCL are multifactorial, but we have shown that arrest of the cell in the G1 phase, due to downregu- lation of E2F, as well as decreased ATP production is Page 10 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Fig. 6 Proposed model of anti-tumor mechanism for metformin in rituximab-sensitive and resistant B-cell lymphoma. Based on our work, we have formulated a model by which we hypothesize metformin induces in its anti-tumor effects. Metformin inhibits proliferation via C-Myc resulting in G1 cell cycle arrest. Metformin causes increased ROS production and loss of mitochondrial membrane potential resulting in apoptosis. Metformin enhances rituximab-mediated ADCC and to a lesser degree CMC Fig. 6 Proposed model of anti-tumor mechanism for metformin in rituximab-sensitive and resistant B-cell lymphoma. Based on our work, we have formulated a model by which we hypothesize metformin induces in its anti-tumor effects. Metformin inhibits proliferation via C-Myc resulting in G1 cell cycle arrest. Metformin causes increased ROS production and loss of mitochondrial membrane potential resulting in apoptos Metformin enhances rituximab-mediated ADCC and to a lesser degree CMC both caused by metformin, and result in decreased cellu- lar proliferation. through cytochrome release and apoptosis [36, 46]. We show here that addition of metformin results in an in- crease in ROS and loss of the mitochondrial membrane permeability in lymphoma cells. Together, these suggest that the increase in apoptosis is mediated by ROS produc- tion resulting in increased membrane permeability. There is likely another factor(s) causing the loss in mitochondrial membrane potential besides increased ROS production, as there was essentially no increase in ROS production in the U2392 cell lines on exposure to metformin. Metformin has been shown to induce apoptosis in leukemia, breast, and esophageal cancer cell lines [43– 45]. We show here that exposure to metformin resulted in dose-dependent increase in apoptosis in both RSCL and RRCL. Prior work by our group has shown that the development of RRCL is associated with a decrease in the pro-apoptotic proteins Bax and/or Bak [4]. These two proteins are responsible for oligomerization and loss of the mitochondrial membrane potential. The findings from Fig. Funding This work was supported, in part, by a grant from the National Cancer Institute (Sponsor Award number 5RO1CA136907-02), a 2012–2013 Develop- mental Funds award from the Roswell Park Alliance Foundation, and a 2016– 2017 Disease Site Research Group (DSRG) Fund award from Roswell Park Alli- ance Foundation and Carl Spezio Lymphoma Research Fund. Supplementary information l f Supplementary information accompanies this paper at https://doi.org/10. 1186/s40170-020-00213-w. Additional file 1: Supplemental Table 1. Descriptive statistics of 16 patient samples treated at Roswell Park Comprehensive Cancer Center. Primary neoplastic B-cells were isolated from pre-treatment biopsy tissue obtained from 16 patients with previously untreated (N=9) or relapsed/re- fractory (N=7) B-cell NHL receiving therapy at Roswell Park Comprehen- sive Cancer Center (RPCCC). Samples from patient biopsy specimens were procured under Institutional Review Board (IRB) RPCCC protocols I42804 and I42904. Tissue specimens were placed in PBS-containing colla- genase type IV (1mg/ml; Sigma-Aldrich, St. Louis, MO) and incubated for 15 minutes at 37°C, followed by manual agitation for five minutes. Next, samples were diluted with RPMI 1640-containing 10% fetal bovine serum (FBS) and the cell suspension filtered through a 100μm cell strainer to re- move large clumps. Lymphocytes were enriched by density centrifuga- tion. B-cells were then isolated from enriched lymphocytes by MACS separation using a human B-cell Isolation Kit II (Miltenyi Biotec, Gladbach, Germany). Abbreviations DLBCL: Diffuse large B-cell lymphoma; RSCL: Rituximab-sensitive cell lines; RRCL: Rituximab-resistant cell lines; ROS: Reactive oxygen species DLBCL: Diffuse large B-cell lymphoma; RSCL: Rituximab-sensitive cell lines; RRCL: Rituximab-resistant cell lines; ROS: Reactive oxygen species Financial/grant support This work was supported, in part, by a grant from the National Cancer Institute (Sponsor Award number 5RO1CA136907-02), a 2012-2013 Develop- mental Funds award from the Roswell Park Alliance Foundation, a 2016-2017 Disease Site Research Group (DSRG) Fund award from Roswell Park Alliance Foundation and Carl Spezio Lymphoma Research Fund Authors’ Contributions SA and JJG contributed equally to this paper; SA, JJG, and FJHI designed the research; SA and JJG analyzed the retrospective clinical study; JJG and QLZ performed the in vitro and in vivo experiments; SA, JJG, and FJHI analyzed the data; SA, JJG, and FJHI wrote the paper. The authors read and approved the final manuscript. Discussion Metformin’s effects on the cell cycle distribu- tion have been shown previously [35–37, 50]. Here, we have also demonstrated that metformin induces G1 cell cycle arrest, with a decrease in the cellular proteins C- Myc, CDK2, E2F, and PCNA. This is confirmed by the decrease in mRNA of C-Myc and PCNA, both of which correspond to the S Phase [51–53]. Of note, our data show that although there was arrest of the cell cycle in G1 for U2392 cell lines, there was minimal effect on C- Myc, PCNA, E2F, or CDK2. The U2392 cell line is de- rived from an ABC DLBCL cell line as opposed to the Raji and RL cell lines, which are derived from GCB DLBCL and BL respectively. It is possible that mechan- ism of cell cycle arrest may be different in the ABC cell lines as opposed to GCB-DLBCL or BL cell lines. perform in laboratory models. It also suggests that by understanding how metformin exercises its anti-tumor effects, a novel inhibitor could be developed with a bet- ter therapeutic range concentration. Additionally, while our work has shown some single agent activity for met- formin, we are not advocating for the use of metformin as a single agent, but rather as chemotherapy enhancer. Conclusion As metformin is becoming more studied for its anti-tumor effects, numerous pre-clinical and clinical studies have also begun focusing on combining metformin with chemother- apy in solid tumor malignancies [19]. Mouse models have shown that metformin plus chemotherapy is more effective than either agent alone [54–56]. We are one of the first groups to report synergistic activity when metformin is combined with either doxorubicin or dexamethasone in lymphoma pre-clinical models. This finding supports the observation noted in our retrospective analysis of DLBCL patients receiving first-line chemotherapy at our Institute. In addition, the cellular effects of metformin can re- sensitize chemotherapy resistant cells to chemo- and im- munotherapy drugs. We are the first to demonstrate here that metformin enhances ADCC of Rituximab in vitro. This effect was seen in both RSCL as well as RRCL and was dose-dependent. To confirm this effect, we studied the ef- fect of adding metformin to rituximab in a lymphoma SCID mice model. Here, we showed that metformin plus rituxi- mab was synergistic in improving the survival of lymphoma bearing SCID mice. This aids in supporting the evaluation of combining metformin with rituximab and/or chemother- apy agents in aggressive B-cell lymphoma clinical trials. Availability of data and materials Our work does have a notable limitation. The concen- tration of metformin in our studies was significantly higher than the typical plasma levels of metformin, which are typically on the order of 10–40 μM [40, 57]. However, in real-world scenarios, patients are typically being exposed constantly to metformin for months to years, potentially leading to broader range in drug blood concentrations. Thus, although they are not exposed to similar plasma concentrations, they are exposed to much longer durations of metformin that are not feasible to All data generated or analyzed during this study are included in this published article and its supplementary information files. Ethics approval and consent to participate pp p p Primary patient samples and retrospective study were approved under Roswell Park Comprehensive Cancer Center Review Board protocols I42804 and I42904; mouse work was reviewed and approved by Roswell Park Comprehensive Cancer Center Animal Care and Use Committee protocol IACUC 966m. Discussion 3a are thus consistent with prior work, show- ing that while metformin results in increased apoptosis, this effect is less pronounced in RRCL, and this may be due to the decreased levels of Bax and/or Bak in these cell lines. Interestingly, even in the RRCL, metformin was able to cause an increase in apoptosis. Cell cycle arrest can affect cancer cell proliferation, be- sides induction of apoptosis. The transition from G1 to S phase is controlled by several growth-dependent cyclin-dependent kinases (CDK) and cyclins which will complex to guide the cell through the cycle. In the early G1 phase, mitogenic stimulation results in synthesis and formation of cyclin D/CDK4/6 complexes. This causes phosphorylation of the RB protein (and their family of proteins). This is a so-called “restriction point,” and after It has been shown previously that excessive mitochon- drial oxidant stress can induce cell death in tumors Page 11 of 13 Page 11 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism this phosphorylation, the cell has committed to progres- sion through from G1 to S phase. Once it is phosphory- lated, pRB dissociates from E2F. This dissociation allows E2F to allow expression of genes for DNA synthesis [47–49]. Metformin’s effects on the cell cycle distribu- tion have been shown previously [35–37, 50]. Here, we have also demonstrated that metformin induces G1 cell cycle arrest, with a decrease in the cellular proteins C- Myc, CDK2, E2F, and PCNA. This is confirmed by the decrease in mRNA of C-Myc and PCNA, both of which correspond to the S Phase [51–53]. Of note, our data show that although there was arrest of the cell cycle in G1 for U2392 cell lines, there was minimal effect on C- Myc, PCNA, E2F, or CDK2. The U2392 cell line is de- rived from an ABC DLBCL cell line as opposed to the Raji and RL cell lines, which are derived from GCB DLBCL and BL respectively. It is possible that mechan- ism of cell cycle arrest may be different in the ABC cell lines as opposed to GCB-DLBCL or BL cell lines. this phosphorylation, the cell has committed to progres- sion through from G1 to S phase. Once it is phosphory- lated, pRB dissociates from E2F. This dissociation allows E2F to allow expression of genes for DNA synthesis [47–49]. References 1. Gisselbrecht C, Glass B, Mounier N, Singh Gill D, Linch DC, Trneny M, et al. Salvage regimens with autologous transplantation for relapsed large B-cell lymphoma in the rituximab era. J Clin Oncol.28(27):4184-90. 1. Gisselbrecht C, Glass B, Mounier N, Singh Gill D, Linch DC, Trneny M, et al. Salvage regimens with autologous transplantation for relapsed large B-cell lymphoma in the rituximab era. J Clin Oncol.28(27):4184-90. 1. Gisselbrecht C, Glass B, Mounier N, Singh Gill D, Linch DC, Trneny M, et al. Salvage regimens with autologous transplantation for relapsed large B-cell lymphoma in the rituximab era. J Clin Oncol.28(27):4184-90. 23. Noto H, Goto A, Tsujimoto T, Noda M. Cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis. PloS one. 2012;7(3):e33411. 2. Thieblemont C, Briere J, Mounier N, Voelker HU, Cuccuini W, Hirchaud E, et al. The germinal center/activated B-cell subclassification has a prognostic impact for response to salvage therapy in relapsed/refractory diffuse large B-cell lymphoma: a bio-CORAL study. J Clin Oncol.29(31):4079-87. 2. Thieblemont C, Briere J, Mounier N, Voelker HU, Cuccuini W, Hirchaud E, et al. The germinal center/activated B-cell subclassification has a prognostic impact for response to salvage therapy in relapsed/refractory diffuse large B-cell lymphoma: a bio-CORAL study. J Clin Oncol.29(31):4079-87. 24. Wynn A, Vacheron A, Zuber J, Solomon SS. Metformin associated with increased survival in type 2 diabetes patients with pancreatic cancer and lymphoma. Am J Med Sci. 2019;358(3):200–3. 25. Wang Y, Maurer MJ, Larson MC, Allmer C, Feldman AL, Bennani NN, et al. Impact of metformin use on the outcomes of newly diagnosed diffuse large B-cell lymphoma and follicular lymphoma. Br J Haematol. 2019;186(6): 820–8. 3. Czuczman MS, Olejniczak S, Gowda A, Kotowski A, Binder A, Kaur H, et al. Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14(5): 1561–70. 26. Fan X ZH, Zhao BB, Yang BS, Zhao Y, Ye J, Lu YM, Wang CF, Xiong H, Chen SJ, Janin A, Wang L, Zhao WL. . Metformin prolonged the survival of diffuse large B-cell lymphoma and grade 3b follicular lymphoma patients responding to first-line treatment with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: a prospective phase II clinical trial. . Transl Cancer Res. 2018;2018;7(4):1044-53. 4. Author details 1 1Texas Oncology, Texas, USA. 2Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, USA. 3Department Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, USA. 4Department of Medical Oncology Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of China. 20. Zhang ZJ, Li S. The prognostic value of metformin for cancer patients with concurrent diabetes: a systematic review and meta-analysis. Diabetes, obesity & metabolism. 2014;16(8):707–10. 21. Gandini S, Puntoni M, Heckman-Stoddard BM, Dunn BK, Ford L, DeCensi A, et al. Metformin and cancer risk and mortality: a systematic review and meta-analysis taking into account biases and confounders. Cancer prevention research (Philadelphia, Pa). 2014;7(9):867-85. Received: 25 October 2019 Accepted: 9 March 2020 References Olejniczak SH, Hernandez-Ilizaliturri FJ, Clements JL, Czuczman MS. Acquired resistance to rituximab is associated with chemotherapy resistance resulting from decreased Bax and Bak expression. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14(5):1550– 60. 27. Cunha Junior AD, Pericole FV, Carvalheira JBC. Metformin and blood cancers. Clinics (Sao Paulo). 2018;73(suppl 1):e412s. 5. Benekli M, Smiley SL, Younis T, Czuczman MS, Hernandez-Ilizaliturri F, Bambach B, et al. Intensive conditioning regimen of etoposide (VP-16), cyclophosphamide and carmustine (VCB) followed by autologous hematopoietic stem cell transplantation for relapsed and refractory Hodgkin’s lymphoma. Bone Marrow Transplant. 2008;41(7):613–9. 28. Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1):275–82 29. 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Received: 25 October 2019 Accepted: 9 March 2020 22. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metformin and reduced risk of cancer in diabetic patients. BMJ (Clinical research ed). 2005;330(7503):1304–5. Consent for publication Not applicable Consent for publication Not applicable Page 12 of 13 Page 12 of 13 Page 12 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. Cancer & Metabolism The authors declare that they have no competing interests. 19. Pierotti MA, Berrino F, Gariboldi M, Melani C, Mogavero A, Negri T, et al. Targeting metabolism for cancer treatment and prevention: metformin, an old drug with multi-faceted effects. Oncogene. 2013;32(12):1475–87. Competing interests 18. Chong CR, Chabner BA. Mysterious metformin. The oncologist. 2009;14(12): 1178–81. g The authors declare that they have no competing interests. References Page 13 of 13 Singh et al. Cancer & Metabolism (2020) 8:10 Singh et al. 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Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by downregulating Stat3 signaling. Cell Death Dis. 2014;5:e1088. 46. Sabharwal SS, Schumacker PT. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles’ heel? Nature reviews Cancer. 2014;14(11):709–21. amplifiers or an Achilles’ heel? Nature reviews Cancer. 2014;14(11):709–21. 47. Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nature reviews Molecular cell biology. 2013;14(8): 518–28. 48. Giacinti C, Giordano A. RB and cell cycle progression. Oncogene. 2006; 25(38):5220–7. 49. Pucci B, Kasten M, Giordano A. Cell cycle and apoptosis. Neoplasia (New York, NY). 2000;2(4):291-9. 50. White-Al Habeeb NM, Garcia J, Fleshner N, Bapat B. Metformin elicits antitumor effects and downregulates the histone methyltransferase multiple myeloma SET domain (MMSET) in prostate cancer cells. The Prostate. 2016; 76(16):1507–18. 51. Hoffman B, Liebermann DA. Apoptotic signaling by c-MYC. Oncogene. 2008; 27(50):6462–72. 52. Naderi S, Wang JYJ, Chen TT, Gutzkow KB, Blomhoff HK. cAMP-mediated inhibition of DNA replication and S phase progression: involvement of Rb, p21(Cip1), and PCNA. Molecular Biology of the Cell. 2005;16(3):1527–42. 53. Yu P, Huang B, Shen M, Lau C, Chan E, Michel J, et al. p15(PAF), a novel PCNA associated factor with increased expression in tumor tissues. Oncogene. 2001;20(4):484–9. 54. Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types. Cancer research. 2011;71(9):3196–201. 55. Lengyel E, Litchfield LM, Mitra AK, Nieman KM, Mukherjee A, Zhang Y, et al. References Metformin inhibits ovarian cancer growth and increases sensitivity to paclitaxel in mouse models. American journal of obstetrics and gynecology. 2015;212(4):479.e1–e10. 56. Yu G, Fang W, Xia T, Chen Y, Gao Y, Jiao X, et al. 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Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27(Kip1 )or p21(Cip1). Journal of Molecular Signaling. 2008;3:18. 15. El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M, Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. The Journal of biological chemistry. 2000; 275(1):223–8. 38. Gui DY, Sullivan LB, Luengo A, Hosios AM, Bush LN, Gitego N, et al. Environment dictates dependence on mitochondrial complex I for NAD+ and aspartate production and determines cancer cell sensitivity to metformin. Cell metabolism. 2016;24(5):716–27. 16. Andrzejewski S, Gravel SP, Pollak M, St-Pierre J. Metformin directly acts on mitochondria to alter cellular bioenergetics. Cancer & metabolism. 2014;2:12. 39. Lossos IS, Morgensztern D. Prognostic biomarkers in diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(6):995–1007. 17. 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https://openalex.org/W2760836924	https://pubs.rsc.org/en/content/articlepdf/2017/sc/c7sc03880a	English	null	Single-cell study of the extracellular matrix effect on cell growth by <i>in situ</i> imaging of gene expression	Chemical science	2,017	cc-by	6,250	Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 This article is licensed under a Creative Commons Attribution 3.0 U Cell behaviors are known to be regulated by the cellular microenvironment. Traditional cell-population based analysis methods need to separate cells from their extracellular matrix (ECM) and cannot resolve the heterogeneity of cell behaviors. Herein, an in situ single-cell analysis method based on rolling circle ampliﬁcation was exploited to image gene expression in single cells for investigating the eﬀect of ECM stiﬀness on cell growth. This method enables the simultaneous quantifying of the cell phenotype and gene expression at the single-cell level, which can help in understanding the underlying molecular mechanism of cell growth. It is found that ECM stiﬀness could aﬀect cell growth via regulating the expression level of the cytoskeleton-assembly associated genes PFN1 and CFL1 and their co-expression pattern. Therefore, this single-cell analysis platform may facilitate us to tap into the study of “single-cell phenotypes” and elucidate the disease association of ECMs. Received 5th September 2017 Accepted 1st October 2017 DOI: 10.1039/c7sc03880a rsc.li/chemical-science Received 5th September 2017 Accepted 1st October 2017 However, the mechanisms of mechanotransduction for cell growth on diﬀerent ECMs remain incompletely understood. Recently, Mooney’s group found that the eﬀect of substrate stress relaxation on cell spreading behavior was mediated through similar pathways as those for substrate stiﬀness: integrin adhesions, Rho activation, actomyosin-based contrac- tility and nuclear translocation of YAP.12 However, Liu’s group investigated the gene expression of cells in response to mechanical stretching. They found that many genes related to cytoskeleton formation greatly changed aer exposure to mechanical stretching (for example, PFN1 and CFL1 increased 13.0 and 1.6 folds, respectively).13 Chemical Science Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China. E-mail: jhli@mail. tsinghua.edu.cn † Electronic supplementary information (ESI) available: Sequence information, in vitro RCA and RT-qPCR data. See DOI: 10.1039/c7sc03880a This journal is © The Royal Society of Chemistry 2017 Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China. E-mail: jhli@mail. tsinghua.edu.cn Single-cell study of the extracellular matrix eﬀect on cell growth by in situ imaging of gene expression† Yupeng Sun, Ruijie Deng, Kaixiang Zhang, Xiaojun Ren, Ling Zhang Cite this: Chem. Sci., 2017, 8, 8019 Yupeng Sun, Ruijie Deng, Kaixiang Zhang, Xiaojun Ren, Ling Zhang d Ji h Li * Yupeng Sun, Ruijie Deng, Kaixiang Zhang, Xiaojun Ren, Ling Zhang and Jinghong Li * † Electronic supplementary information (ESI) available: Sequence information, in vitro RCA and RT-qPCR data. See DOI: 10.1039/c7sc03880a Introduction The extracellular matrix (ECM) not only provides a physical support for cell adhesion but also serves an important instructional role, providing biochemical and biomechanical cues.1–3 Specically, the stiﬀness of the ECM plays an important role in regulating cell behaviors such as cell spreading, migra- tion, proliferation and diﬀerentiation.4–8 Cells sense their ECM stiﬀness through a mechanotransduction signaling pathway which is a cellular process that translates external mechanical stimuli into intracellular biochemical signals.3,9 Cell growth and functions are regulated by gene expression programs and the disturbance of gene expression can result in many human diseases. The occurrence of cancer is not only caused by the activation of proto-oncogene and deregulation of cell-cycle control, but also abnormal defective mechano- transduction signaling may lead to tumor formation and metastatic progression.10 For example, a disturbance in ECM mechanics stimulates the Rho-ROCK-MLC pathway, increases cytoskeletal tension, completes a self-enforcing (positive) feed- back loop and results in further increases in ECM stiﬀness, which can promote malignant transformation, tumorigenesis and metastasis.11 Recently, pillar arrays,14 traction force microscopy15 and atomic force microscopy (AFM)16,17 have been successfully applied to in situ determine cellular traction forces exerted by the interaction of cells and their ECMs by measuring the pillar displacement or the substrate deformations. However, detailed descriptions of the molecular mechanisms are still missed due to the lack of genetic information. Previous attempts to char- acterize gene or protein expression programs and investigate the molecular mechanisms were based on methods such as quantitative PCR, western blotting or RNA sequencing. Never- theless, cells must be isolated from their cultured substrates when nucleic acids are extracted, resulting in cell–substrate interactions missed and cell status changes. Meanwhile, the overall average data lack the information of cell heterogeneity, which is widespread in biological systems, and may lead to inaccurate results.18–20 Herein, we have developed an in situ single-cell mRNA imaging method to investigate the eﬀect of extracellular matrix Chem. Sci., 2017, 8, 8019–8024 \| 8019 This journal is © The Royal Society of Chemistry 2017 Edge Article View Article Online Chemical Science shown in Fig. 1A. Briey, a padlock probe was designed to recognize the target sequence of mRNA, then specically ligate and circularize with the mRNA as the template. n Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Firstly, to demonstrate the feasibility of rolling circle ampli- cation for RNA detection, the target sequence of ACTB mRNA was amplied in vitro. According to the results of the uorescence spectra and gel electrophoresis characterization, the padlock probe can specically recognize and eﬀectively amplify the target sequence by RCA (Fig. S1–S3†). We further tested the perfor- mance for imaging RNAs in situ in single cells. As illustrated in Fig. 1B, the bright dots amplied from the target mRNA are easily distinguished from the background. A control experiment was conducted without a trigger primer which was used to initiate DNA polymerization and no obvious uorescence signal was observed (Fig. 1C), which conrmed that the bright dots resulted from in situ RCA. Next, to verify the specicity of this method, a random padlock probe was used and no distinct uorescence signal was observed (Fig. 1D). Furthermore, a blocked probe was designed to prevent the padlock probe from binding with the target sequence. Few bright dots could be detected (Fig. 1E), suggesting that the bright dot signals came from the target mRNA. The ACTB expression ranged from approximately 84 to at most 200 copy number per cell (the average was 115 per cell) and presented a normal distribution (Fig. 1B, inset; Fig. S4† shows how to quantify the copy number). The obvious variability sug- gested that even the same batch cells would exhibit signicant cell-to-cell variation in gene expression. Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In situ RCA for the analysis of cytoskeleton related gene expression in single cells Scheme 1 illustrates the eﬀect of extracellular matrix stiﬀness on cell growth and the molecular mechanisms investigated via an in situ single-cell platform. As illustrated in Scheme 1A, the cells present diﬀerent shapes when cultured on soand stiﬀ substrates, and the cells on the stiﬀsubstrate show a spindle shape and obvious stress bers. A robust RNA imaging method based on in situ rolling circle amplication (RCA) has been developed to investigate the mRNA expression in single cells at the single-molecule level.21,22 Scheme 1B illustrates the proposed mechanisms of how the stiﬀness of the ECM aﬀects the cell behaviors. Briey, cells sense the ECM stiﬀness via an integrin triggered mechanotransduction pathway and the increased ECM stiﬀness induces specic gene expression related to cytoskeletal rearrangement (PFN1 and CFL1), forms actin laments and promotes cell spreading and growth.23,24 Introduction Next, the target mRNA could be amplied by RCA, resulting in a long DNA molecule with a large number of repeat sequences.26–28 Upon hybridization with the detection probes, the RCA amplicon would become visible as a diﬀraction-limited uorescent spot. stiﬀness on cell growth. In this method, the relationships of single-cell gene expressions, morphology phenotype and the eﬀect of diﬀerent ECMs were investigated by simultaneous in situ imaging of the cell morphology and mRNA without a complicated pretreatment process for the cells. The expres- sion of cytoskeleton related mRNA (PFN1, CFL1 and ACTB) for cells cultured on diﬀerent substrates was visualized at single- molecule levels. The multi-parameter, in situ single-cell study of the extracellular matrix eﬀect on cell growth indicates the complexity and heterogeneity of cell behaviors responding to diﬀerent ECMs. Timescale of gene expression during cell growth It’s a complicated process for cells to sense the ECM stiﬀness via a mechanotransduction pathway which includes sensing, signalling and gene expression to lead cell function. The time- scale of these events ranges from seconds to weeks.3 To inves- tigate the timescale of cell–substrate interaction, we utilized our single-cell mRNA imaging method to visualize the gene expression of cells in diﬀerent culture times. As shown in Fig. 3A, the cells present varied morphologies and mRNA expression at diﬀerent time points. It was found that the quantity of ACTB mRNA increased with the incubation time and reached a maximum (120 copies per cell on average) aer 10 h attachment (Fig. 3B). This may be because the cell spreading process was nished and the cells tended to express less ACTB mRNA.33,34 Besides, the coeﬃcient of variation of ACTB mRNA copy numbers per cell was more than 0.2 (Fig. 3B), indicating that the degree of dispersion for ACTB mRNA expression in diﬀerent cells was high. These results emphasize the impor- tance of single-cell mRNA detection due to cell heterogeneity. As the incubation time increased, the ACTB mRNA uctuations evolved from a narrow and peaked histogram into a widely dispersed prole, with the average shiing to higher copy numbers (Fig. 3C). The single-cell proling results indicate that the distribution pattern of gene expressions in single cells can be distinct at diﬀerent stages and may help us to understand the procedure of cell spreading and the formation of stress bers. Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Next, to quantify the parameters related to cell morphology, the cell spreading area, aspect ratio (AR) and circularity were taken into statistical analysis. Typically, the cells on the so substrate (1 kPa) present a round shape and smaller spreading area (400 mm2), while the cells cultured on the stiﬀsubstrate (30 kPa) show a spindle shape and larger spreading area (1600 mm2). As presented in Fig. 2B, the cell spreading area increased monotonically with substrate stiﬀness. Meanwhile, the aspect ratio and cell circularity (as descriptors of cell shape) were acquired from cell images using Image J. Evaluation of cell behaviors on diﬀerent stiﬀness substrates Sci., 2017, 8, 8019–8024 View Article Online Edge Article Chemical Science coated polyacrylamide (PAAm) gels with diﬀerent elastic moduli.29 The hydrogel stiﬀness was controlled by the ratio and the concentration of acrylamide and bis-acrylamide (details in ESI Table 1†) and the surface morphology of the hydrogel substrate is similar (Fig. S5 and S6†). The cells were cultured on gels for 12 h, then xed and stained with Alexa 488-conjugated phalloidin and DAPI to reveal the actin lament network (green) and the nuclei (blue), respectively. The representative uores- cence images of the MCF-7 cells are shown in Fig. 2A. Obvious stress bers (lamentous actin bundles) were seen in cells grown on a stiﬀer substrate (30 kPa) and a glass substrate (50 GPa), but not in cells grown on sosubstrates (1 kPa, 4 kPa, and 13 kPa). Stress bers, which play an important role in cellular cytoskeleton formation, can provide force for cells to sense and transmit the signal of the substrate stiﬀness and help cells to spread and grow.30 suggests that ECM stiﬀness can aﬀect the formation of stress bers, cell shape and spreading. Timescale of gene expression during cell growth The aspect ratio of the cells was calculated as the ratio between the cell length and width.31 We found that the aspect ratio of the cells increased with substrate stiﬀness, reaching a plateau (at about 1.5) on the 13 kPa PAAm gels. Circularity, which reects the roundness of the cells, is dened as the spreading area multi- plied by 4p and divided by the square of the perimeter.32 Similarly, the circularity decreased with increasing substrate stiﬀness, reaching a plateau (at approximately 0.7) on the 13 kPa PAAm gels. The distributions of these statistical parameters are presented in Fig. S7.† Therefore, the morphological analysis Gene expression variation with ECM stiﬀness To investigate the eﬀect of substrate stiﬀness on cell gene expression at the single-cell level, the expression of Fig. 3 Cell morphology and ACTB mRNA expression for diﬀerent culture times. (A) Imaging of cell morphology (bright ﬁeld) and ACTB mRNA by in situ RCA (dark ﬁeld). Scale bars, 25 mm. (B) The average expression level and coeﬃcient of variation for ACTB mRNA with diﬀerent incubation times. (C) Single-cell ﬂuctuation proﬁles for ACTB mRNA with diﬀerent incubation times (n > 100, C.V. ¼ s.d./mean). Fig. 2 Cell morphologies on diﬀerent stiﬀness substrates and quan- titative analysis. (A) Fluorescence images of MCF-7 cells on PAAm gels with varied stiﬀness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins (green) and nuclei (blue) after culturing on the substrate for 12 h. Scale bar, 25 mm. Quantitative analysis of the cell morphology on the substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n > 100; mean s.d.; *P < 0.05, P < 0.01, NS, not signiﬁcant, compare to glass). Fig. 3 Cell morphology and ACTB mRNA expression for diﬀerent culture times. (A) Imaging of cell morphology (bright ﬁeld) and ACTB mRNA by in situ RCA (dark ﬁeld). Scale bars, 25 mm. (B) The average expression level and coeﬃcient of variation for ACTB mRNA with diﬀerent incubation times. (C) Single-cell ﬂuctuation proﬁles for ACTB mRNA with diﬀerent incubation times (n > 100, C.V. ¼ s.d./mean). This journal is © The Royal Society of Chemistry 2017 Chem. Sci., 2017, 8, 8019–8024 \| 8021 Fig. 2 Cell morphologies on diﬀerent stiﬀness substrates and quan- titative analysis. (A) Fluorescence images of MCF-7 cells on PAAm gels with varied stiﬀness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins (green) and nuclei (blue) after culturing on the substrate for 12 h. Scale bar, 25 mm. Quantitative analysis of the cell morphology on the substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n > 100; mean s.d.; *P < 0.05, P < 0.01, NS, not signiﬁcant, compare to glass). Fig. 2 Cell morphologies on diﬀerent stiﬀness substrates and quan- titative analysis. Evaluation of cell behaviors on diﬀerent stiﬀness substrates ACTB is a constitutive housekeeping gene which plays a critical role in F-actin formation (generating the actin monomer as the basic building unit of F-actin).25 A scheme of in situ RCA is To investigate the inuence of ECM stiﬀness on cell morphology, MCF-7 cells were cultured on diﬀerent collagen- Fig. 1 In situ imaging of the expression of ACTB mRNA in single cells. (A) The scheme of in situ RCA in single cells. Imaging of ACTB mRNA by RCA using the target padlock probe (B), without the trigger primer (C), using a random padlock probe (D), and after blocking the target site (E). The cell nuclei are stained by DAPI (blue), the RCA amplicons are hybridized with the Alexa 488-ﬂuorescence probe (green spots), and the cell outlines are marked by a dotted line. Inset: frequency histo- grams of RCA amplicons per cell detected. Scale bars, 20 mm. Scheme 1 An illustration of the mechanism of cell growth on the extracellular matrix with varied stiﬀness via an in situ single-cell plat- form. (A) The eﬀect of extracellular matrix stiﬀness on cell growth and the procedures of in situ RCA for detecting gene expression in single cells. (B) The proposed mechanisms of how the stiﬀness of the ECM aﬀects the cell growth and gene expression. Fig. 1 In situ imaging of the expression of ACTB mRNA in single cells. (A) The scheme of in situ RCA in single cells. Imaging of ACTB mRNA by RCA using the target padlock probe (B), without the trigger primer (C), using a random padlock probe (D), and after blocking the target site (E). The cell nuclei are stained by DAPI (blue), the RCA amplicons are hybridized with the Alexa 488-ﬂuorescence probe (green spots), and the cell outlines are marked by a dotted line. Inset: frequency histo- grams of RCA amplicons per cell detected. Scale bars, 20 mm. Scheme 1 An illustration of the mechanism of cell growth on the extracellular matrix with varied stiﬀness via an in situ single-cell plat- form. (A) The eﬀect of extracellular matrix stiﬀness on cell growth and the procedures of in situ RCA for detecting gene expression in single cells. (B) The proposed mechanisms of how the stiﬀness of the ECM aﬀects the cell growth and gene expression. This journal is © The Royal Society of Chemistry 2017 8020 \| Chem. Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 Gene co-expression patterns and correlation analysis. (A) PFN1–CFL1 expression correlation analysis in a single cell on diﬀerent substrates. (B) The relationship of the single-cell spreading area and gene co-expression pattern (PFN1/CFL1) (n > 100, mean s.d.; P < 0.05, ‘r’ means the correlation coeﬃcient of the single-cell gene co- expression of PFN1 and CFL1). expression variations of PFN1 and CFL1, rather than ACTB, play more important roles in the cell spreading behavior when cultured on substrates with diﬀerent degrees of stiﬀness. As the cell spreading process involves the rapid formation of the new actin laments and the degradation of the remaining laments, it demands the up-regulation of the gene expression of PFN1 and CFL1. The increased expression level of colin (encoded by CFL1) would facilitate the depolymerization of the old actin laments into actin monomers and prolin (encoded by PFN1) can elongate new actin laments with the depolymerized actin monomers to facilitate the cell spreading.37 To infer the regulatory connections between single-cell behavior and gene expression, the single-cell gene expression and cell spreading area were in situ investigated simultaneously and quantitatively. A correlation coeﬃcient is regarded as a main parameter that quanties a type of correlation and dependence. The correlation coeﬃcient between the cell spreading area and the copy number on the stiﬀsubstrate for genes ACTB, PFN1, and CFL1 was 0.486, 0.580 and 0.375, and 0.482, 0.370 and 0.475 on the sosubstrate, respectively (Fig. 4B). Compared to the sosubstrate, the correlation coef- cient between the cell spreading area and gene PFN1 increased and decreased for CFL1 on the stiﬀsubstrate, while it remained constant for ACTB. The single-cell correlations of the cell spreading area and gene expression indicated that the p g Cell growth is a complicated process involving multiple genes, and we hypothesize that gene co-expression patterns have signicant inuences on cell behaviors on diﬀerent ECMs. To test this hypothesis, we measured the single-cell co- expression patterns of key genes related to cytoskeleton rear- rangement. The single-cell correlation coeﬃcients of PFN1 and CFL1 on a stiﬀsubstrate and sosubstrate were 0.741 and 0.736, respectively (Fig. 5A). The high correlation coeﬃcients indicated that this gene pair could be regulated by a common upstream gene or directly regulate each other.38 Besides, it was found that the average ratios of PFN1 and CFL1 (PFN1/CFL1) in single cells were 2.570 and 1.747 for cells cultured on stiﬀand sosubstrates, respectively (Fig. 5B). Gene expression variation with ECM stiﬀness (A) Fluorescence images of MCF-7 cells on PAAm gels with varied stiﬀness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins (green) and nuclei (blue) after culturing on the substrate for 12 h. Scale bar, 25 mm. Quantitative analysis of the cell morphology on the substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n > 100; mean s.d.; P < 0.05, P < 0.01, NS, not signiﬁcant, compare to glass). Fig. 2 Cell morphologies on diﬀerent stiﬀness substrates and quan- titative analysis. (A) Fluorescence images of MCF-7 cells on PAAm gels with varied stiﬀness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins (green) and nuclei (blue) after culturing on the substrate for 12 h. Scale bar, 25 mm. Quantitative analysis of the cell morphology on the substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n > 100; mean s.d.; P < 0.05, P < 0.01, NS, not signiﬁcant, compare to glass). Fig. 2 Cell morphologies on diﬀerent stiﬀness substrates and quan- titative analysis. (A) Fluorescence images of MCF-7 cells on PAAm gels with varied stiﬀness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins (green) and nuclei (blue) after culturing on the substrate for 12 h. Scale bar, 25 mm. Quantitative analysis of the cell morphology on the substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n > 100; mean s.d.; *P < 0.05, P < 0.01, NS, not signiﬁcant, compare to glass). Fig. 3 Cell morphology and ACTB mRNA expression for diﬀerent culture times. (A) Imaging of cell morphology (bright ﬁeld) and ACTB mRNA by in situ RCA (dark ﬁeld). Scale bars, 25 mm. (B) The average expression level and coeﬃcient of variation for ACTB mRNA with diﬀerent incubation times. (C) Single-cell ﬂuctuation proﬁles for ACTB mRNA with diﬀerent incubation times (n > 100, C.V. ¼ s.d./mean). This journal is © The Royal Society of Chemistry 2017 Chem. Sci., 2017, 8, 8019–8024 \| 8021 Fig. 5 Gene co-expression patterns and correlation analysis. (A) PFN1–CFL1 expression correlation analysis in a single cell on diﬀerent substrates. Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. These data indicated that gene co-expression patterns could be regulated by substrate stiﬀness. A possible mechanism for cell growth is that ACTB is a structural gene, and PFN1 and CFL1 are regulatory genes involved in stress ber formation, enhancing cell spreading and promoting cell growth. It is worth emphasizing that not only the expression level of PFN1 and CFL1 but also the gene co- expression patterns can regulate the assembling of the cyto- skeleton in the cell spreading process. Fig. 4 Correlation analysis of the single-cell spreading area and gene expression. (A) Images of single-cell spreading behavior and gene expression of ACTB, PFN1 and CFL1 on diﬀerent stiﬀness substrates (4 kPa and 30 kPa). Scale bars, 25 mm. Inset: frequency histograms of RCA amplicons per cell detected. (B) The relationship of single-cell gene expression and cell spreading area for ACTB, PFN1 and CFL1, respectively (n > 100, mean s.d.; P < 0.05, ‘r’ means the correlation coeﬃcient of the single-cell spreading area and copy number per cell). Gene expression variation with ECM stiﬀness (B) The relationship of the single-cell spreading area and gene co-expression pattern (PFN1/CFL1) (n > 100, mean s.d.; P < 0.05, ‘r’ means the correlation coeﬃcient of the single-cell gene co- expression of PFN1 and CFL1). Edge Article View Article Online Chemical Science cytoskeleton-related genes (ACTB, PFN1 and CFL1) in cells cultured on substrates with varying stiﬀness was in situ detec- ted. These genes play important roles in cytoskeleton remod- eling which is a signicant process for cell growth and metastasis.35 The spreading area on the sosubstrate was restricted to around 600 mm2, while on the stiﬀsubstrate it reached around 1600 mm2 (Fig. 4A). Specically, for the cells cultured on the stiﬀsubstrate, the expression of PFN1 and CFL1 was nearly two times higher than for the cells cultured on the soer substrate. However, the ACTB mRNA expression level was almost consistent (1.11 fold increase compared to the so substrate). Generally, actins (ACTB) can be used repeatedly as the basic building units of F-actin in stress ber forming and cytoskeleton remodeling, therefore the ACTB mRNA expression level remains basically unchanged.36 As a validation, we compared the mRNA expression of ACTB, PFN1 and CFL1 averaged over hundreds of cells by in situ RCA to those obtained from a bulk RNA quantitative measurement (RT-qPCR) per- formed on the same cell line. Our imaging results are in good accordance with the RT-qPCR results in general (Fig. S8†). Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6:43:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Conclusions Fig. 4 Correlation analysis of the single-cell spreading area and gene expression. (A) Images of single-cell spreading behavior and gene expression of ACTB, PFN1 and CFL1 on diﬀerent stiﬀness substrates (4 kPa and 30 kPa). Scale bars, 25 mm. Inset: frequency histograms of RCA amplicons per cell detected. (B) The relationship of single-cell gene expression and cell spreading area for ACTB, PFN1 and CFL1, respectively (n > 100, mean s.d.; **P < 0.05, ‘r’ means the correlation coeﬃcient of the single-cell spreading area and copy number per cell). In summary, we report an in situ single-cell imaging method for investigating the eﬀect of extracellular matrix stiﬀness on cell growth. In this method, there is no need to lyse large numbers of cells to acquire enough RNA, or to separate cells from the cultured substrate. This method can yield a simultaneous and This journal is © The Royal Society of Chemistry 2017 8022 \| Chem. Sci., 2017, 8, 8019–8024 Chemical Science View Article Online Chemical Science View Article Online Edge Article 365 nm ultraviolet light for 10 min, washed twice with 50 mM HEPES in PBS and then incubated in 200 mg mL1 of rat type I collagen in HEPES overnight at 37 C. detailed description of the single-cell gene expression proles and morphology variance under the physical microenviron- ments. It is found that the increase of PFN1 and CFL1 mRNA expression levels and change in the PFN1 and CFL1 co- expression pattern, which is triggered by the stiﬀsubstrates, greatly promote the assembling of the cytoskeleton in the cell growth process. Therefore, the in situ mRNA imaging method can help us to understand the molecular mechanism for the inuence of ECM mechanical cues on cell growth and metas- tasis at a deeper level. Moreover, it provides the potential to understand the mechanisms of ECM defect related diseases. Cell staining and image analysis The MCF-7 cells on the substrates were xed with 4% PFA for 10 min and permeabilized with 0.5% Triton-X100 for 5 min at room temperature, and then blocked with 1% BSA for 1 h for actin lament staining. Actin staining was performed using Alexa-Fluor 488 conjugated to phalloidin (Life Technologies, UK). Aer post-stain washing with PBS, the cells were mounted in 4,6-diamidino-2-5-phenylindole (DAPI, Vector Laboratories, USA) for nuclear staining. For measurements of the cell- spreading area in 2D, images of the phalloidin/DAPI-stained cells were taken using a Leica TCS SP5 inverted confocal microscope (Leica, Germany) with a 63 oil-immersion objec- tive. Only those cells that did not exhibit any cell–cell contacts were considered in the analysis. Images of all single cells were then thresholded manually on the basis of the actin stain, and the cell spreading area was determined using Image J soware. In situ visualization of mRNAs in a single-cell by RCA As in a typical in situ RCA detection experiment, the hybridiza- tion of the target mRNA with the padlock probe was carried out in a volume of 20 mL solution, produced by adding 2 mL padlock probe (10 mM), 1 mL DTT (100 mM), 0.5 mL RiboLock RNase Inhibitor (40 U mL1) and 4 mL yeast tRNA (10 mg mL1) to 12.5 mL RNase-free water, overnight at 37 C. Then, the sample was washed using PBS-T (DEPC-PBS with 0.05% Tween-20) three times at room temperature. The ligation process was conducted in a volume of 10 mL containing 1 mL T4 DNA ligase (5 U mL1), 1 mL 10 T4 DNA ligase reaction buﬀer, 0.25 mL RiboLock RNase Inhibitor (40 U mL1) and RNase-free water at 37 C for 2 h. The primer hybridization reaction was then per- formed with a 20 mL mixture containing 1 mL primer (5 mM), 1 mL DTT (100 mM), 2 mL 20 SSC, 2 mL formamide, 13.5 mL RNase- free water and 0.5 mL RiboLock RNase Inhibitor (40 U mL1) for 60 min at 37 C. The RCA reaction mixture containing 1 mL 10 phi29 DNA polymerase reaction buﬀer, 0.5 mL phi29 DNA Preparation of polyacrylamide hydrogels Briey, PAAm gel solutions containing acrylamide monomers, cross-linker bis-acrylamide, ammonium persulphate and tetra- methylethylenediamine (TEMED) were prepared. The ratio of acrylamide and bis-acrylamide and the nal concentrations were varied to control the hydrogel stiﬀness and porosity (details in ESI Table 1†). Glass coverslips were activated by Piranha solution (H2SO4 : H2O2 ¼ 3 : 1) and then functionalized using 3-(trimethoxysilyl)propyl methacrylate (APTES) and glutaraldehyde to facilitate the covalent attachment of the hydrogel substrates to the amino-silanated coverslips. The gel solution was sandwiched between the functionalized coverslip and a chloro-silanated glass slide to ensure easy detachment of the hydrogels. Materials and apparatus All synthetic oligonucleotides (Table S3†) were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). The RCA detection probes were modied with Alexa488 and Cy5, and were purchased from Thermo Fisher Scientic (Waltham, USA). The salmon sperm DNA, 20 SSC buﬀer (pH 7.4) and 4% paraformaldehyde in PBS buﬀer were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The deoxyribonucleotides mixture (dNTPs) was purchased from Beijing DingGuo Biotechnology Co., Ltd. (Beijing, China). Tween-20, diethy pyrocarbonate (DEPC), formamide, Triton-X100 and 3-amino- propyl triethoxysilane were purchased from Sigma-Aldrich (St. Louis, USA). TransScript one-step gDNA removal and cDNA synthesis were purchased from Transgen Biotech Co., Ltd. (Beijing, China). RiboLock RNase Inhibitor, T4 polynucleotide kinase, T4 DNA ligase, phi29 DNA polymerase, RevertAid First Strand cDNA Synthesis Kit and SYBR select master mix were purchased from Thermo Fisher Scientic (Waltham, USA). All of the solutions and deionized water used were treated with DEPC and autoclaved to be protected from RNase degradation. Cell culture The MCF-7 cells were cultured in standard Dulbecco’s Modied Eagle’s medium with 10% fetal bovine serum, 1% penicillin/ streptomycin, and 0.01 mg mL1 human recombinant insulin. The cells were incubated at 37 C, 5% CO2 and 95% air humidity. Cells were seeded on 22 22 mm collagen-coated glass coverslips (VWR, Radnor, USA) enclosed with PDMS as a chamber (5 mm in diameter). The varying stiﬀness hydrogel substrates modied with collagen were washed three times with PBS and placed in the cell culture hood for 30 min under UV light for sterilization before cell seeding. For cell seeding, the cells were plated at a proper density onto the hydrogel substrates with diﬀerent degrees of stiﬀness, so that they had enough space to spread and didn’t contact other cells. Cells were allowed to spread for 12 h, and then were xed and stained for analysis. Acknowledgements 21 R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin and J. Li, Angew.Chem., Int. Ed., 2014, 53, 2389–2393. This work was nancially supported by the National Natural Science Foundation of China (No. 21621003, No. 21235004, No. 21327806), and the Tsinghua University Initiative Scientic Research Program. 22 R. Deng, K. Zhang and J. Li, Acc. Chem. Res., 2017, 50(4), 1059–1068. 23 P. P. Provenzano and P. J. Keely, J. Cell Sci., 2011, 124, 1195– 1205. 24 K. Xu, G. Zhong and X. Zhuang, Science, 2013, 339, 452–456. Open Access Article. Published on 02 October 2017. Downloaded on 10/24/2024 6: This article is licensed under a Creative Commons Attribution 3.0 Unp 20 R. A. Burrell, N. McGranahan, J. 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Tang, Gene, 2012, 501, 153–163. 8024 \| Chem. Sci., 2017, 8, 8019–8024 This journal is © The Royal Society of Chemistry 2017
https://openalex.org/W4361969663	https://figshare.com/articles/journal_contribution/Figure_S5_from_Local_Intracerebral_Immunomodulation_Using_Interleukin-Expressing_Mesenchymal_Stem_Cells_in_Glioblastoma/22474896/1/files/39926394.pdf	Dutch	null	Figure S4 from Local Intracerebral Immunomodulation Using Interleukin-Expressing Mesenchymal Stem Cells in Glioblastoma	null	2,023	cc-by	171	Supplemental Figure 5 A B GL261 bearing mice Spleen weight day 15 MSC injection at day 10 PBS MSC-GFP MSC-IL 0.05 0.06 0.07 0.08 0.09 0.10 0.11 Spleen weight [g] * ** Spleen weight [g] PBS MSC-GFP MSC-IL7/12 d5 MSC-IL7/12 d10 0.04 0.05 0.06 0.07 0.08 0.09 0.052 0.055 CT-2A bearing mice Spleen weight at endpoint Supplemental Figure 5 A B GL261 bearing mice Spleen weight day 15 MSC injection at day 10 PBS MSC-GFP MSC-IL 0.05 0.06 0.07 0.08 0.09 0.10 0.11 Spleen weight [g] * ** Spleen weight [g] PBS MSC-GFP MSC-IL7/12 d5 MSC-IL7/12 d10 0.04 0.05 0.06 0.07 0.08 0.09 0.052 0.055 CT-2A bearing mice Spleen weight at endpoint Supplemental Figure 5 g B Spleen weight [g] PBS MSC-GFP MSC-IL7/12 d5 MSC-IL7/12 d10 0.04 0.05 0.06 0.07 0.08 0.09 0.052 0.055 CT-2A bearing mice Spleen weight at endpoint A GL261 bearing mice Spleen weight day 15 MSC injection at day 10 PBS MSC-GFP MSC-IL 0.05 0.06 0.07 0.08 0.09 0.10 0.11 Spleen weight [g] * ** B A
https://openalex.org/W3082627327	https://www.frontiersin.org/articles/10.3389/fnut.2020.565996/pdf	English	null	Biochemical and Nutritional Evaluation of Chlorella and Auxenochlorella Biomasses Relevant for Food Application	Frontiers in nutrition	2,020	cc-by	8,098	Edited by: Lilia Ahrné, University of Copenhagen, Denmark Edited by: Lilia Ahrné, University of Copenhagen, Denmark Reviewed by: Chaogang Wang, Shenzhen University, China Juan E. Andrade, University of Florida, United States Correspondence: Alexander Mathys alexander.mathys@hest.ethz.ch Reviewed by: Chaogang Wang, Shenzhen University, China Juan E. Andrade, University of Florida, United States Correspondence: Alexander Mathys alexander.mathys@hest.ethz.ch Specialty section: This article was submitted to Nutrition and Food Science Technology, a section of the journal Frontiers in Nutrition Biochemical and Nutritional Evaluation of Chlorella and Auxenochlorella Biomasses Relevant for Food Application Greta Canelli 1, Carmen Tarnutzer 1, Roberta Carpine 2,3, Lukas Neutsch 2, Christoph J. Bolten 4, Fabiola Dionisi 4 and Alexander Mathys 1* Microalgae are a source of potentially healthy and sustainable nutrients. However, the bioaccessibility of these nutrients remains uncertain. In this study, we analyzed the biomass composition of ﬁve commercial Chlorella and Auxenochlorella strains, and Chlorella vulgaris heterotrophically cultivated in our laboratory. Protein accounted for 65 ± 3% (w w−1) dry matter (DM) in all biomasses, except for the lab-grown C. vulgaris that contained 20% (w w−1) DM protein. The fatty acids content was comparable and ranged between 7 and 10% (w w−1) DM. Most of the biomasses had a ω6-polyunsaturated fatty acids (PUFAs)/ω3-PUFAs ratio < 4, as recommended by nutritional experts. A recently published harmonized protocol for in vitro digestion was used to evaluate fatty acids and protein bioaccessibilities. Protein bioaccessibility ranged between 60 and 74% for commercial Chlorella and Auxenochlorella biomasses and was 43% for the lab-grown C. vulgaris. Fatty acids bioaccessibility was < 7% in commercial biomasses and 19% in the lab-grown C. vulgaris. Taken together, the results show that microalgae are promising sources of bioaccessible protein. The limited fatty acids bioaccessibility indicates the need for alternative upstream and downstream production strategies. Keywords: microalgae, Chlorella, Auxenochlorella, bioaccessibility, omega-3-polyunsaturated fatty acids, protein, digestion INTRODUCTION Microalgal biomass is an emerging sustainable source of proteins, fatty acids, carotenoids, and carbohydrates with potential health beneﬁts for humans (1–4). Moreover, microalgae often have essential amino acids composition meeting FAO requirements, and they are frequently on par with other protein sources, such as soybean and egg (5). Chlorella spp. together with Arthrospira spp. (known as “Spirulina”) account for the largest production volume (6). These two genera are among the few that are allowed to be consumed as whole biomass in Europe according to European Union food regulations (7). However, their biomass composition can vary considerably. Knowledge of the biochemical proﬁle of the biomass is necessary for the selection of appropriate microalgae for speciﬁc food applications. Diﬀerent biomass compositions for the same species, or even strains, are reported in the literature. This variability is partially due to diﬀerences in cultivation conditions. Factors that include Received: 26 May 2020 Accepted: 17 August 2020 Published: 30 September 2020 ORIGINAL RESEARCH published: 30 September 2020 doi: 10.3389/fnut.2020.565996 Citation: Canelli G, Tarnutzer C, Carpine R, Neutsch L, Bolten CJ, Dionisi F and Mathys A (2020) Biochemical and Nutritional Evaluation of Chlorella and Auxenochlorella Biomasses Relevant for Food Application. Front. Nutr. 7:565996. doi: 10 3389/fnut 2020 565996 September 2020 \| Volume 7 \| Article 565996 Frontiers in Nutrition \| www.frontiersin.org Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. pH, temperature, salinity, and nutrient availability can aﬀect the biomass composition (2, 8). The variation that has been described might also be attributed to the diﬀerent analytical methods used (9). When measuring nitrogen to calculate protein content, species-speciﬁc nitrogen-to-protein conversion factors should be used, instead of the standard 6.25. Even though conversion factors may vary depending on growth stage and cultivation conditions, a conversion factor of 6.35 was proposed for Chlorella vulgaris (10). In view of this inconsistency, there is need for studies comparing biomass proﬁles using standardized protocols. pH, temperature, salinity, and nutrient availability can aﬀect the biomass composition (2, 8). The variation that has been described might also be attributed to the diﬀerent analytical methods used (9). When measuring nitrogen to calculate protein content, species-speciﬁc nitrogen-to-protein conversion factors should be used, instead of the standard 6.25. Even though conversion factors may vary depending on growth stage and cultivation conditions, a conversion factor of 6.35 was proposed for Chlorella vulgaris (10). In view of this inconsistency, there is need for studies comparing biomass proﬁles using standardized protocols. Limited literature is available on Chlorella and Auxenochlorella nutrient bioaccessibilities. For commercial Chlorella biomasses, an average protein digestibility of 51 ± 9% was reported (4). In a previous work, we studied the bioaccessibility of fatty acids by an infant in vitro digestion model in C. vulgaris, which was limited to 3% (23). To date, no study on fatty acids bioaccessibility in adults for Chlorella or Auxenochlorella has been published. Considering this knowledge gap, this study evaluated the bioaccessibilities of fatty acids and proteins in several Chlorella and Auxenochlorella biomasses relevant for food applications using a harmonized protocol. In addition, biochemical composition and nutritional parameters were assessed following standardized procedures. Commercially available biomasses were compared to C. vulgaris heterotrophically grown in our laboratory. From a nutritional perspective, microalgae present interesting proﬁles rich in several nutritional and health-beneﬁcial components, such as polyunsaturated fatty acids (PUFAs). Citation: In particular, ω3-PUFAs such as α-linolenic acid, are essential fatty acids that must be supplied in the diet, as they cannot be synthesized by the human body (11). Several important indices, termed indices of lipid nutritional quality (INQ), need to be considered when evaluating the fatty acids proﬁle of an ingredient. Nutritional experts have recommended a ω6-PUFAs/ω3-PUFAs ratio < 4 as desirable (12–14). Previous studies reported that a ω6/ω3 ratio < 4 reduced total mortality by 70% in the prevention of cardiovascular diseases, a ratio of 5 was beneﬁcial for asthma, a ratio of 2–3 reduced rheumatoid arthritis inﬂammation, and a ratio of 2.5 reduced colorectal cancer cell proliferation (15). There is no general indication on the recommended ω6/ω3 ratio provided by the European Food Safety Authority. However, several European countries established their own recommendations (16). The German- Austrian-Swiss recommendations, as well as the Nutritional Recommendations for the French Population, recommend a ω6/ω3 ratio of 5. The Nordic Nutrition Recommendations considers a ω6/ω3 ratio between 3 and 9 to be adequate (16). The consumption of foods rich in ω3-PUFAs in Western countries is limited and often scarce (17). Therefore, microalgae can be a promising alternative/supplementation to oil sources, such as ﬁsh and plant-based sources (18). MATERIALS AND METHODS Acquisition of Algal Biomass Frontiers in Nutrition \| www.frontiersin.org Acquisition of Algal Biomass Five commercially available dried Chlorella and Auxenochlorella biomasses were purchased: Alver “Golden Chlorella,” Biotona, Piura, Purasana, and Soleil Vie (Table 1). For each biomass, genus, brand, supplier, place and date of purchase, country of origin, treatment after harvest, and species are reported. Additionally, C. vulgaris biomass was heterotrophically produced in our laboratory as previously described (23). C. vulgaris (CCALA 256) was obtained from the Culture Collection of Autotrophic Organisms in the Czech Republic. Batch cultivation was performed in a 16-L laboratory bioreactor (Bilﬁnger Industrial Technologies, Salzburg, Austria) with a working volume of 10 L. The temperature was set at 28 ◦C, the stirring speed was 300 rpm, the dissolved oxygen tension was kept above 75%, and aeration (4 L min−1) was achieved with ﬁlter-sterilized air. The pH was kept constant at 7 by automatic addition of H2SO4 (0.5 M) and NaOH (0.5 M). The medium used for growth was modiﬁed BBM (nitrate concentration of 1.5 g L−1) enriched with 15 g L−1 glucose. The culture was harvested after 7 days of growth, frozen, and freeze-dried for further analysis. Nutrients, such as protein and fatty acids, are present in microalgae, which are surrounded by a cell wall. It is hypothesized that microalgae cell wall, being mainly composed by indigestible polysaccharides, cannot be degraded by the digestive enzymes present in the mouth, stomach, and small intestine (19). This would limit the nutrient digestibility and bioaccessibility, which has not been thoroughly investigated for many important compounds found in microalgae. Bioaccessibility is deﬁned as the fraction of a food/component that is released from the food matrix in the gastrointestinal tract that becomes available for absorption (20). Bioaccessibility is often determined by in vitro digestion. Even though in vitro methods strongly simplify reality, they have some advantages over in vivo methods. Generally, in vitro methods are more rapid, less expensive, less labor intensive, do not have ethical restrictions, and are very suitable for mechanistic studies and hypothesis building (21). However, dissimilar protocols are often used for the assessment. The use of a common protocol for in vitro digestion is essential for data comparison. In this study, we followed the recently published harmonized INFOGEST 2.0 protocol (22). Microalgae Biomass Composition (1) Atherogenicity index (AI) = [(C12:0 + (4 × C14:0) + C16:0)]/(MUFAs + Ñ6-PUFAs + Ñ3-PUFAs) (27) (1) Atherogenicity index (AI) = [(C12:0 + (4 × C14:0) + C16:0)]/(MUFAs + Ñ6-PUFAs + Ñ3-PUFAs) (27) (2) Thrombogenicity index (TI) = (C14:0 + C16:0 + C18:0)/[(0.5 × MUFAs) + (0.5 × Ñ6-PUFAs) + (3 × Ñ3-PUFAs) + (Ñ3-PUFAs/Ñ6-PUFAs)] (27) (2) Thrombogenicity index (TI) = (C14:0 + C16:0 + C18:0)/[(0.5 × MUFAs) + (0.5 × Ñ6-PUFAs) + (3 × Ñ3-PUFAs) + (Ñ3-PUFAs/Ñ6-PUFAs)] (27) (3) Hypocholesterolemic/hypercholesterolemic fatty acids ratio (H/H) = (C18:1-Ñ9 + C18:2-Ñ6 + C 18:3-Ñ3)/(C14:0 + C16:0) (28) (4) P/S = PUFAs/SFAs (4) P/S = PUFAs/SFAs (4) P/S = PUFAs/SFAs (5) ω6/ω3 = Ñ6-PUFAs/Ñ3-PUFAs. (5) ω6/ω3 = Ñ6-PUFAs/Ñ3-PUFAs. Microalgae Biomass Composition In brief, fatty acids in freeze-dried biomass were directly trans-esteriﬁed using 1.5 N methanolic hydrochloric acid solution and analyzed by gas chromatography using an instrument equipped with a split-injection port and ﬂame ionization detection (FID) (7890 A; Agilent Technologies, Basel, Switzerland). The following temperature–time program was used: 50 ◦C (0.2 min), 50–180 ◦C (120 ◦C min−1), 180– 220 ◦C (6.7 ◦C min−1), and 220–250 ◦C (30 ◦C min−1) on a 70% cyanopropyl polysilphenylene-siloxane column with a length of 10 m, internal diameter of 0.1 mm, and ﬁlm of 0.2 µm (BPX70; SGE Analytical Science, Milton Keynes, UK). Peak identiﬁcation was performed by comparing the retention times with FAME standards (Nu-Chek Prep. Inc., Elysian, USA). The peak areas were quantiﬁed with OpenLab CDS VL software (Agilent Technologies, Basel, Switzerland). of saturated fatty acids (SFAs, C12:0, C14:0, C16:0, and C18:0), monounsaturated fatty acids (MUFAs, C15:1-Ñ5, C16:1-Ñ7, C17:1-Ñ7, C18:1-Ñ9), and polyunsaturated fatty acids (PUFAs, C18:2-Ñ6, C18:3-Ñ3) according to: (LECO Corporation, St. Joseph, MI, USA). Protein content was estimated from the total nitrogen content, multiplied by an overall conversion factor of 6.35, as previously proposed for C. vulgaris (10). Fatty acids proﬁle of the biomass was determined as previously reported (23). In brief, fatty acids in freeze-dried biomass were directly trans-esteriﬁed using 1.5 N methanolic hydrochloric acid solution and analyzed by gas chromatography using an instrument equipped with a split-injection port and ﬂame ionization detection (FID) (7890 A; Agilent Technologies, Basel, Switzerland). The following temperature–time program was used: 50 ◦C (0.2 min), 50–180 ◦C (120 ◦C min−1), 180– 220 ◦C (6.7 ◦C min−1), and 220–250 ◦C (30 ◦C min−1) on a 70% cyanopropyl polysilphenylene-siloxane column with a length of 10 m, internal diameter of 0.1 mm, and ﬁlm of 0.2 µm (BPX70; SGE Analytical Science, Milton Keynes, UK). Peak identiﬁcation was performed by comparing the retention times with FAME standards (Nu-Chek Prep. Inc., Elysian, USA). The peak areas were quantiﬁed with OpenLab CDS VL software (Agilent Technologies, Basel, Switzerland). Microalgae Biomass Composition Purasana Purasana NV, Gullegem, Belgium Apo24.ch, Switzerland, 2019 Mongolia or Hainan 30.11.2021 Cell walls broken by an unknown treatment Chlorella vulgaris Soleil Vie Montasell SA, La Tour-de-Trême, Switzerland Coop, Switzerland, 2019 China 26.10.2020 n. a. Chlorella vulgaris Auxenochlorella Alver “Golden Chlorella” Golden Chlorella SA, Chardonne, Switzerland Alver.ch, Switzerland, 2018 n. a. n. a. n. a. Auxenochlorella protothecoides (24) FIGURE 1 \| Schematic representation of the in vitro digestion protocol applied on biomass suspensions (SSF, simulated salivary ﬂuid; SGF, simulated gastric ﬂuid; SIF, simulated intestinal ﬂuid). TABLE 1 \| Overview of the powdered Chlorella and Auxenochlorella biomasses. Genus Brand Supplier Place and year of purchase Country of origin Expiry date Treatment after harvest according to the supplier Species Chlorella Biotona KeyPharm, Oostkamp, Belgium Apo24.ch, Switzerland, 2019 China 03.2022 Dehydrated by a superior drying process Chlorella pyrenoidosa Piura Green Origins, Shefﬁeld, Great Britain Narayana Verlag, Germany, 2019 Asia 09.2021 Cell walls broken by an high-impact jet spray process before drying and milling Chlorella spp. Purasana Purasana NV, Gullegem, Belgium Apo24.ch, Switzerland, 2019 Mongolia or Hainan 30.11.2021 Cell walls broken by an unknown treatment Chlorella vulgaris Soleil Vie Montasell SA, La Tour-de-Trême, Switzerland Coop, Switzerland, 2019 China 26.10.2020 n. a. Chlorella vulgaris Auxenochlorella Alver “Golden Chlorella” Golden Chlorella SA, Chardonne, Switzerland Alver.ch, Switzerland, 2018 n. a. n. a. n. a. Auxenochlorella protothecoides (24) TABLE 1 \| Overview of the powdered Chlorella and Auxenochlorella biomasses. FIGURE 1 \| Schematic representation of the in vitro digestion protocol applied on biomass suspensions (SSF, simulated salivary ﬂuid; SGF, simulated gastric ﬂuid; SIF, simulated intestinal ﬂuid). FIGURE 1 \| Schematic representation of the in vitro digestion protocol applied on biomass suspensions (SSF, simulated salivary ﬂuid; SGF, simulated gastric ﬂuid; SIF, simulated intestinal ﬂuid). of saturated fatty acids (SFAs, C12:0, C14:0, C16:0, and C18:0), monounsaturated fatty acids (MUFAs, C15:1-Ñ5, C16:1-Ñ7, C17:1-Ñ7, C18:1-Ñ9), and polyunsaturated fatty acids (PUFAs, C18:2-Ñ6, C18:3-Ñ3) according to: of saturated fatty acids (SFAs, C12:0, C14:0, C16:0, and C18:0), monounsaturated fatty acids (MUFAs, C15:1-Ñ5, C16:1-Ñ7, C17:1-Ñ7, C18:1-Ñ9), and polyunsaturated fatty acids (PUFAs, C18:2-Ñ6, C18:3-Ñ3) according to: (LECO Corporation, St. Joseph, MI, USA). Protein content was estimated from the total nitrogen content, multiplied by an overall conversion factor of 6.35, as previously proposed for C. vulgaris (10). Fatty acids proﬁle of the biomass was determined as previously reported (23). Microalgae Biomass Composition Microalgae Biomass Composition For each microalgae species, triplicate samples of dried biomass were analyzed to determine moisture, carbohydrate, protein, fatty acids contents, and fatty acids composition. Moisture content was determined by weighing before and after drying 1.3 ± 0.3 g of biomass for 24 h at 80 ◦C. The average moisture content was used in the calculation of the biochemical composition, and was expressed as percentage of total dry matter (DM). Carbohydrate content was determined by the anthrone method as previously described (25). This method is commonly used for quantitative measurement of total carbohydrates in microalgae because of its high sensitivity and simplicity (26). Protein content was determined by the Dumas method. Approximately 0.5 g of biomass was transferred to ceramic crucibles and total nitrogen content was analyzed using a TruMac CN device September 2020 \| Volume 7 \| Article 565996 Frontiers in Nutrition \| www.frontiersin.org 2 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. TABLE 1 \| Overview of the powdered Chlorella and Auxenochlorella biomasses. Genus Brand Supplier Place and year of purchase Country of origin Expiry date Treatment after harvest according to the supplier Species Chlorella Biotona KeyPharm, Oostkamp, Belgium Apo24.ch, Switzerland, 2019 China 03.2022 Dehydrated by a superior drying process Chlorella pyrenoidosa Piura Green Origins, Shefﬁeld, Great Britain Narayana Verlag, Germany, 2019 Asia 09.2021 Cell walls broken by an high-impact jet spray process before drying and milling Chlorella spp. Purasana Purasana NV, Gullegem, Belgium Apo24.ch, Switzerland, 2019 Mongolia or Hainan 30.11.2021 Cell walls broken by an unknown treatment Chlorella vulgaris Soleil Vie Montasell SA, La Tour-de-Trême, Switzerland Coop, Switzerland, 2019 China 26.10.2020 n. a. Chlorella vulgaris Auxenochlorella Alver “Golden Chlorella” Golden Chlorella SA, Chardonne, Switzerland Alver.ch, Switzerland, 2018 n. a. n. a. n. a. Auxenochlorella protothecoides (24) FIGURE 1 \| Schematic representation of the in vitro digestion protocol applied on biomass suspensions (SSF, simulated salivary ﬂuid; SGF, simulated gastric ﬂuid; SIF, simulated intestinal ﬂuid). TABLE 1 \| Overview of the powdered Chlorella and Auxenochlorella biomasses. Genus Brand Supplier Place and year of purchase Country of origin Expiry date Treatment after harvest according to the supplier Species Chlorella Biotona KeyPharm, Oostkamp, Belgium Apo24.ch, Switzerland, 2019 China 03.2022 Dehydrated by a superior drying process Chlorella pyrenoidosa Piura Green Origins, Shefﬁeld, Great Britain Narayana Verlag, Germany, 2019 Asia 09.2021 Cell walls broken by an high-impact jet spray process before drying and milling Chlorella spp. Determination of Fatty Acids and Protein Bioaccessibilities Protein and fatty acids bioaccessibilities were determined by an in vitro digestion model (Figure 1), according to the standardized protocol (INFOGEST 2.0) (22). Simulated salivary ﬂuid (SSF), simulated gastric ﬂuid (SGF), and simulated intestinal ﬂuid (SIF) were prepared exactly as recommended by Brodkorb et al. (22). Indexes of Lipid Nutritional Quality (INQ) The nutritional quality of the lipid fraction was assessed by ﬁve separate indexes. These are calculated based on the concentration September 2020 \| Volume 7 \| Article 565996 Frontiers in Nutrition \| www.frontiersin.org 3 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. TABLE 2 \| Macronutrient composition (% per 100 g DM) of dried microalgal biomasses. Biotona Piura Purasana Soleil Vie Alver LG-Chlorella % dry matter Carbohydrates EV 11.7 ± 1.7 14.1 ± 0.7 11.0 ± 0.2 9.9 ± 0.4 20.2 ± 0.2 65.0 ± 0.3 PV 29 17.3 22 5.2 23.5 Proteins EV 63.4 ± 0.0 62.7 ± 0.1 65.5 ± 0.1 64.1 ± 0.0 59.6 ± 0.0 18.9 ± 0.0 PV 58 59.1 60 59.1 63 Fatty acids EV 9.8 ± 0.4 9.7 ± 0.6 9.2 ± 1.0 9.0 ± 0.6 10.0 ± 1.2 8.0 ± 0.1 PV 12 13.4 15 13.4 11 Experimental results are expressed as mean ± standard deviation (n = 3) and compared to reference values on the packaging’s label. EV, experimental value; PV, packaging value. TABLE 2 \| Macronutrient composition (% per 100 g DM) of dried microalgal biomasses. In brief, the digestion was performed in amber glass in a water bath at 37 ◦C and stirring set at 300 rpm. The oral phase (2 min, pH 7) started with mixing of the biomass (1 g) with water (3.78 mL), SSF (3.2 mL), and CaCl2 (20 µL, 0.3 M). To simulate the gastric phase, the oral bolus was then mixed with SGF (6.4 mL) and CaCl2 (4 µL, 0.3 M), and the pH adjusted to 3. Pepsin (0.4 mL, 80,000 U mL−1; Sigma-Aldrich, Buchs, Switzerland) and gastric lipase (0.4 mL, 2,400 U mL−1; Lipolytech, Marseille, France) were added and the total volume was adjusted to 16 mL with water. The pH was constantly adjusted to 3. After 2 h of incubation with stirring, the pH was adjusted to 7 to simulate the intestinal phase and SIF (6.8 mL), CaCl2 (32 µL, 0.3 M), pancreatin (4 mL, 800 U mL−1; Sigma- Aldrich), and bile salts (2 mL, 0.16 mM; Sigma-Aldrich) were added. Water was added to a total volume of 32 mL. During 2 h incubation with stirring, pH was constantly adjusted to 7. As blank, digestion without biomass was performed. At the end of the intestinal phase, an aliquot (6 mL) of full digesta was snap-frozen with liquid nitrogen and freeze-dried. Data Analysis Data were shown as the mean ± standard deviation of three independent replicates (n = 3). All statistical analyses in the present study were performed using Rstudio software (v4.0.0). The assumptions for parametric tests (equal variance and normality) were tested by Levene’s test and Shapiro-Wilk test, respectively, for each factor. When the assumptions were met, one-way ANOVA combined with Tukey’s test for multiple comparison was performed to assess statistical signiﬁcance (p < 0.05). For all other factors, a non-parametric tests (Kruskal- Wallis) followed by the Dunn’s Multiple Comparison test (p < 0.05) was performed. The results of statistical analysis are reported in the Supplementary Material (Table S1). Indexes of Lipid Nutritional Quality (INQ) The residue was centrifuged (30 min, 10,000 × g, 4 ◦C). The micellar phase (supernatant) and the pellet were individually snap-frozen and freeze-dried. As a positive control, infant formula (Aptamil 1; Milupa, Dublin, Ireland) was also subjected to in vitro digestion, as complete bioaccessibility was expected for this sample. As a blank, 4 mL of water without microalgal biomass was digested, in order to quantify the fatty acids and nitrogen coming from the enzymes and digestive ﬂuids. This is further referred to as enzyme blank. in equation 1. (1) Frontiers in Nutrition \| www.frontiersin.org Biochemical Characterization of Microalgal Biomasses Five commercial microalgal biomasses (Biotona, Piura, Purasana, Soleil Vie and Alver) and a biomass of C. vulgaris heterotrophically grown in our laboratory (hereafter termed LG-Chlorella) were characterized for their macronutrient composition. Results are expressed as percentage on dry matter, as they were corrected for the moisture contents (3.5–5.9%) (Table 2). Fatty acids and protein contents were measured in the micellar phase, pellet, and full digesta. Total fatty acids were measured as explained in section Microalgae Biomass Composition. Protein content was determined by total nitrogen measurement using a TOC-L equipped with a TN module (Shimadzu Europa, Duisburg, Germany). The dried micellar phase and full digesta (10–20 mg) were dissolved in 15 mL of water and analyzed. Due to poor solubility, the dried pellet was measured using the Dumas method as explained in section Microalgae Biomass Composition. Fatty acids/protein bioaccessibility was deﬁned as the amount of fatty acids/protein incorporated into the micellar phase (corrected by the fatty acids/protein in the micellar phase of enzyme blank) compared the amount of fatty acids/protein in the full digesta (corrected by the fatty acids/protein in the full digesta of enzyme blank), as expressed in percentage (%) The carbohydrate content in Biotona biomass was 12%, which was lower than the previously reported value of 26% for C. pyrenoidosa biomass (1). Purasana and Soleil Vie biomasses (both C. vulgaris), contained 10–11% carbohydrate, which was comparable to previously reported values of 10–17% for this species (1, 9). Higher carbohydrate contents were observed for Piura (14%) and Alver (20%), which consist of Chlorella spp. and Auxenochlorella protothecoides (24), respectively. The yellow color of Alver may suggest that the biomass was grown in heterotrophic conditions (29), which could explain the higher carbohydrate content (30). LG-Chlorella that was cultivated September 2020 \| Volume 7 \| Article 565996 4 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. TABLE 3 \| Fatty acid proﬁle of commercial microalgae biomasses and LG-Chlorella. Biotona Piura Purasana Soleil Vie Alver LG-Chlorella 10:0 n.d. n.d. 0.2 ± 0.3 n.d. n.d. n.d. 14:0 n.d. n.d. 0.1 ± 0.2 n.d. 1.8 ± 0.0 n.d. 15:1-ω5 1.3 ± 0.0 1.2 ± 0.0 1.2 ± 0.0 1.0 ± 0.7 n.d. n.d. 16:0 18.2 ± 0.1 18.7 ± 0.1 18.1 ± 0.3 19.6 ± 1.0 15.9 ± 0.0 20.2 ± 0.0 16:1-ω7 2.6 ± 0.1 1.0 ± 0.0 1.2 ± 0.0 2.7 ± 0.1 n.d. n.d. Fatty Acid Composition The fatty acid composition was analyzed by identifying the main fatty acids, as well as the proportion of total SFAs, monounsaturated fatty acids (MUFAs), and PUFAs. Ten fatty acids, ranging from of C10:0 to 18:3-ω3, were identiﬁed and quantiﬁed as the percentage of the total fatty acid content of the algal biomasses (Table 3). The three most abundant fatty acids in all biomasses were palmitic (16:0), linoleic (18:2-ω6), and α-linolenic (18:3-ω3) acids, with values ranging 20–25%, 35– 50%, and 10–20%, respectively. Exceptionally, Alver contained a higher amount of oleic acid (40%) than the other biomasses (3–15%), and lower amounts of α-linolenic acid (10%), palmitic acid (16%), and linoleic acid (31%). The residual fatty acids identiﬁed (10:0, 14:0, 15:1-ω5, 16:1-ω7, 17:1-ω7, 18:0, and 18:1- ω9) accounted for 10% or less of total fatty acids (with the exception of 18:1-ω9 for LG-Chlorella). No EPA and DHA were detected in any biomass. All biomasses had a similar fatty acids proﬁle, except for Alver, which could be attributed to the diﬀerent genera (24, 29). Compared to other biomasses, Alver displayed lower amounts of SFAs and PUFAs, but higher MUFAs content. The total fatty acids content was similar for all biomasses and ranged between 9 and 10%. These ﬁndings are consistent with that of Muys et al. (4). The authors reported an average of 7.5% fatty acids for several commercial Chlorella biomasses. In general, C. vulgaris can reach fatty acids values between 5 and 40% under optimal growth conditions for cell growth and proliferation and up to 58% under unfavorable conditions (33, 34). High variability in the fatty acids content (6–58%) was also reported for A. protothecoides, the species with which Alver was identiﬁed (24). Experimental results were compared with the information on the packaging provided by the supplier. The comparison between package labeling and analysis revealed several diﬀerences. The experimental carbohydrate content deviated from what was reported on the labels, with the exception of Alver. This could be explained because carbohydrates on the nutritional label are often calculated by subtracting moisture, protein, fatty acids, and ash content from 100% (35). We observed higher protein and lower fatty acids values for all the biomasses, with the exception of the Alver biomass. Our results conﬁrmed Alver’s declaration. Overall, the diﬀerence between the supplier’s information and experimental results could be explained by the use of diﬀerent analytical methods. Frontiers in Nutrition \| www.frontiersin.org Biochemical Characterization of Microalgal Biomasses 17:1-ω7 7.8 ± 0.1 4.8 ± 0.1 6.4 ± 0.1 8.7 ± 0.4 n.d. n.d. 18:0 2.3 ± 0.0 2.7 ± 0.0 2.7 ± 0.0 2.7 ± 0.2 1.6 ± 0.0 4.9 ± 0.0 18:1-ω9 2.8 ± 0.0 2.7 ± 0.0 2.1 ± 0.0 2.6 ± 0.1 38.9 ± 0.2 14.8 ± 0.0 18:2-ω6 27.7 ± 0.0 37.5 ± 0.0 34.1 ± 0.5 31.1 ± 1.6 30.6 ± 0.1 40.3 ± 0.1 18:3-ω3 15.1 ± 0.3 7.6 ± 0.1 10.1 ± 0.1 15.8 ± 0.8 9.4 ± 0.1 19.7 ± 0.0 OFAs 22.2 ± 0.1 23.8 ± 0.3 23.9 ± 0.5 15.8 ± 4.6 1.8 ± 0.3 n.d. 6 SFAs 20.5 ± 0.1 21.4 ± 0.1 21.2 ± 0.7 22.3 ± 1.1 19.3 ± 0.1 25.1 ± 0.0 6 MUFAs 14.5 ± 0.2 9.7 ± 0.2 10.8 ± 0.2 15.0 ± 1.4 38.9 ± 0.2 14.8 ± 0.0 6 PUFAs 42.8 ± 0.3 45.1 ± 0.2 44.1 ± 0.6 46.9 ± 2.4 40.0 ± 0.2 60.0 ± 0.1 Fatty acids are expressed as percentage (%) of the total fatty acids. Results are expressed as mean of triplicates ± standard deviation (n = 3). OFA, Other fatty acid; SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid; n.d., not detected. TABLE 3 \| Fatty acid proﬁle of commercial microalgae biomasses and LG-Chlorella. probably measured the total fat content gravimetrically upon ether extraction, which leads to inclusion of other compounds than fatty acids (36). under heterotrophic conditions in the presence of glucose had the highest carbohydrate value of 65%. Protein content was around 60–66% for all biomasses, except LG-Chlorella, indicating their potential as protein sources. LG- Chlorella contained 20% protein. The diﬀerence might be explained by diﬀerent culture conditions. Nitrogen repletion promotes growth and protein production, whereas nitrogen limitation or depletion retards growth and reduces protein content, but favors starch and/or fat accumulation in cells (23, 31, 32). LG-Chlorella was harvested after 4 days of nitrogen limitation, which could explain the high carbohydrate and low protein contents. Fatty Acid Composition We expressed the fat content as the sum of the total measured fatty acids. Diﬀerently, the vendors Diﬀerent fatty acid proﬁles for C. vulgaris have been reported (37–39). Lower contents of SFAs were detected in all biomasses (19–25%), compared to the value of 33.5% previously reported by Batista et al. (37). The same study reported a higher proportion of MUFAs (24.9%) instead of the 10–15% in the Chlorella biomasses we analyzed. Biomasses of Biotona, Piura, Purasana, Soleil Vie, and Alver had PUFAs contents of 40–47%, which agreed with that detected by Batista et al. (37). LG-Chlorella contained 60% PUFAs, indicating its exceptional potential for application in the development of healthy food products. September 2020 \| Volume 7 \| Article 565996 5 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. TABLE 4 \| Nutritional quality indexes of the lipid fraction of the analyzed microalgal biomasses. Biotona Piura Purasana Soleil Vie Alver LG-Chlorella P/S 2.09 ± 0.01 2.11 ± 0.01 2.08 ± 0.05 2.11 ± 0.01 2.08 ± 0.01 2.39 ± 0.01 ω6/ω3 1.84 ± 0.04 4.93 ± 0.11 3.40 ± 0.06 1.97 ± 0.01 3.27 ± 0.02 2.05 ± 0.00 H/H 2.51 ± 0.01 2.56 ± 0.01 2.53 ± 0.03 2.53 ± 0.02 4.47 ± 0.03 3.70 ± 0.01 AI 0.32 ± 0.00 0.34 ± 0.00 0.34 ± 0.02 0.32 ± 0.00 0.29 ± 0.00 0.27 ± 0.00 TI 0.31 ± 0.00 0.46 ± 0.00 0.40 ± 0.01 0.32 ± 0.00 0.31 ± 0.00 0.29 ± 0.00 Results are expressed as the mean of triplicates ± standard deviation (n = 3). P/S, polyunsaturated/saturated; ω6/ω3, Ñ6-PUFAs/Ñ3-PUFAs; H/H, hypocholesterolemic/hypercholesterolemic fatty acids ratio; AI, atherogenicity index; TI, thrombogenicity index. TABLE 4 \| Nutritional quality indexes of the lipid fraction of the analyzed microalgal biomasses. FIGURE 2 \| Fatty acids and protein bioaccessibility (%) in microalgae biomasses, expressed as mean of digestion triplicates (n = 3) ± standard deviation. reported by Matos et al. (28). Alver and LG-Chlorella had even higher H/H values of 4.5 and 3.7, respectively. The H/H values in microalgae were lower compared to that of chia (H/H = 11.4) or ﬂax seeds (H/H = 17.3) (40, 41). When compared to marine ﬁsh (H/H = 0.9–2.5), the analyzed microalgal biomass had an excellent H/H ratio (42). According to Ulbricht and Southgate (27), the atherogenicity index (AI), and thrombogenicity index (TI) evaluate the potential for stimulating platelet aggregation. Frontiers in Nutrition \| www.frontiersin.org Fatty Acid Composition There are no recommended values for AI and TI. The lower the AI and TI values, the higher the protective potential against heart coronary diseases (40). In addition, recent studies found positive associations between both general and abdominal obesity and AI and TI (43). Furthermore, a positive association between gestational diabetes mellitus and TI was reported in pregnant women (44). Myristic acid (C14:0) and palmitic acid (C16:0) are among the most atherogenic agents, whereas stearic acid (C18:0) is considered thrombogenic but not atherogenic (45). In this study, AI values ranged between 0.27 and 0.34, while TI was 0.29–0.46. The lowest AI and TI of 0.27 and 0.29, respectively, were found in LG-Chlorella. These values agreed very well with the data reported for marine ﬁsh, where AI = 0.26–0.60 and TI = 0.20–0.44 (42). Chia (AI = 0.09, TI = 0.05) and ﬂax seeds (AI = 0.06, TI = 71.7) presented lower AI and TI, except for a much higher TI in ﬂax seeds (40, 41). FIGURE 2 \| Fatty acids and protein bioaccessibility (%) in microalgae biomasses, expressed as mean of digestion triplicates (n = 3) ± standard deviation. Indexes of Lipid Nutritional Quality (INQ) CONCLUSIONS The study determined the biochemical composition of Chlorella and Auxenochlorella biomasses. Protein accounted for 65 ± 3% in all biomasses, except for the lab-grown C. vulgaris that contained 20% protein. The fatty acids content was comparable and ranged between 7 and 10%. All biomasses showed a relevant fat nutritional quality, with balanced ω6/ω3, P/S, H/H, AI, and TI indexes. The protein bioaccessibility was > 40% for all biomasses, while the fatty acids bioaccessibility was < 7% in commercial biomasses and 19% in LG-Chlorella. Taken together, the results show that microalgae are promising sources of bioaccessible protein. Regarding fatty acids, their limited bioaccessibility indicates the need for alternative upstream and downstream production strategies. In case of fatty acids bioaccessibility, LG-Chlorella showed the highest value (19%) compared to the commercially available biomasses (< 7%). No relevant variation was observed among the commercial biomasses for fatty acids bioaccessibility. Additionally, we calculated the bioaccessibilities of single fatty acids. Results showed that there was no signiﬁcant diﬀerence in the bioaccessibilities between the type of fatty acids (data not shown). Comparable data for fatty acids bioaccessibility in the literature are scarce. Alternatively, data on bioaccessibility of β- carotene and lutein, which are associated with the fat droplets, are available (34). Gille et al. (48) reported that no β-carotene and only 7% of lutein was bioaccessible in C. vulgaris biomass (48). Our results are similar. In addition, they explained the higher bioaccessibility of lutein by its higher hydrophilicity and, therefore, higher release in the aqueous micellar phase compared to β-carotene. Interestingly, protein bioaccessibility values are clearly distinct from the fat bioaccessibility values. Saﬁet al. (49) reported that C. vulgaris proteins are almost all hydro-soluble, which could explain the simpler diﬀusion of those in the aqueous micellar phase during digestion. Moreover, protein localization within the algae might facilitate their bioaccessibility, with 20% cell wall associated, 30% able to diﬀuse freely, and 50% located internally (50). It might be that the intracellular and the freely diﬀusible proteins are more bioaccessible. Contrarily, the very limited fatty acids bioaccessibility may indicate that fatty acids are not able to easily diﬀuse out of the cell wall, contact digestive enzymes, and become incorporated in the micellar phase (51). In support of our hypothesis, Zhang et al. (52) showed that some of the fatty acids in Chlorella are attached to the cell wall, probably linked to carbohydrates by an ether bond. ACKNOWLEDGMENTS The authors gratefully acknowledge the Nestlé Research, Lausanne, Switzerland, and the ETH Zurich Foundation, Switzerland, for their support. In addition, the authors sincerely thank Sabrina Tevere, from the Institute of Chemistry and Biotechnology, ZHAW, Campus Grüental, Wädenswil, Switzerland, for support with the biomass cultivation. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. CONCLUSIONS Another reason for the limited fat bioaccessibility could be that free fatty acids that are liberated after hydrolysis have established complexes with proteins/carbohydrates or salts (such as calcium) and precipitated in the pellet obtained by centrifugation after digestion (21). ndexes of Lipid Nutritional Quality (INQ) H/H for Biotona, Piura, Purasana, and Soleil Vie were ∼2.5, which was higher than the value of 2 September 2020 \| Volume 7 \| Article 565996 6 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. data, because information about disruption treatment of the biomass from the supplier were unclear. digestibility of the chitin-like cell wall, for which humans do not possess digestive enzymes. Overall, the protein bioaccessibility observed in this study is comparable to the 51 ± 9% reported by Muys et al. (4) for commercial Chlorella biomasses. The deviation of our results might reﬂect the diﬀerent in vitro digestion protocol used. We used the latest version of the standardized INFOGEST protocol (22), whereas Muys et al. (4) followed the ﬁrst version, which did not yet include gastric lipase (21). Although gastric lipase is more relevant for fat digestion, there is always an interplay between any digestive enzyme and nutrient bioaccessibility. Other authors found protein digestibility values between 27 and 70% for Chlorella, but followed diﬀerent protocols (46, 47). The variability found in data reported in literature highlights the importance of using a harmonized protocol. Further research should explore the nutrient bioaccessibilities of microalgae biomass upon in vivo digestion, to overcome the limitations of in vitro studies, such as the absence of physiological adaptation in pH and enzyme concentration, interaction with the gut microbiota, and mechanical dynamics (22). Moreover, it would be valuable to investigate nutritional and biochemical qualities of additional microalgae species interesting for the food and nutraceutical industries. AUTHOR CONTRIBUTIONS GC and CT: methodology, formal analysis, and writing—original draft. GC and AM: project administration. GC, RC, LN, CB, FD, and AM: conceptualization, writing—review, and editing. AM: funding acquisition. All authors contributed to the article and approved the submitted version. ndexes of Lipid Nutritional Quality (INQ) Indexes of Lipid Nutritional Quality (INQ) The nutritional quality of the lipid proﬁles of the analyzed biomasses was evaluated by ﬁve diﬀerent indexes (Table 4), as previously described (28). A polyunsaturated-to-saturated fatty acids (P/S) ratio < 0.45 is considered undesirable in food, because of the potential to induce an increase in blood cholesterol (17). The P/S of the analyzed microalgae biomasses exceeded 2. From a nutritional perspective, a balanced ω6/ω3 fatty acids ratio is important for the prevention and management of obesity, as well as for the reduction of the risk of chronic diseases (13, 15). Typical western diets show preponderance of ω6 over ω3 fatty acids, mainly due to the greater consumption of ω-6 rich vegetable oils (e.g., sunﬂower, peanut, corn) compared to ω-3 sources, such as ﬁsh and nuts (15). All investigated biomasses showed a ω6/ω3 ratio < 5. In particular, Biotona, Soleil Vie, and LG-Chlorella reported an ideal ratio between 1 and 2. Fatty Acids and Protein Bioaccessibilities Fatty acids and protein bioaccessibilities in the analyzed biomasses are reported in Figure 2. The validity of the digestion model was tested by including a positive control (infant formula), which showed a protein and fatty acids bioaccessibility of 97.5 ± 3.2% and 88.7 ± 4.5%, respectively. The protein bioaccessibility was 60, 63, 74, and 43% for Biotona, Piura, Alver, and LG- Chlorella, respectively. Alver had a higher protein bioaccessibility than Biotona and LG-Chlorella. The latter had the lowest protein bioaccessibility of all biomasses. A high polysaccharide content is one of the main factors that negatively inﬂuences protein digestibility, as reviewed by Bleakley and Hayes (5). This could explain the lower protein bioaccessibility of LG-Chlorella, which had a carbohydrate content of 70%. Piura was reported to have cell walls broken by a high-impact jet spray process before drying and milling. Alver and Biotona biomasses were likely to be disrupted as they had a similar or even a higher protein bioaccessibility than Piura. 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Microalgae characterization for consolidated and new application in human food, animal feed and nutraceuticals. Int J Environ Res Public Health. (2018) 15:1–21. doi: 10.3390/ijerph15112436 17. FAO/WHO. Fats and Fatty Acids in Human Nutrition— Report of an Expert Consultation. Geneva: FAO/WHO (2008). 18. Matos J, Cardoso C, Bandarra NM, Afonso C. Microalgae as healthy ingredients for functional food: a review. Food Funct. (2017) 8:2672– 85. doi: 10.1039/C7FO00409E 39. Becker WE. Microalgae for human animal nutrition. In: Amos R, Hu Q, editors. Handbook of Microalgal Culture: Applied Phycology Biotechnology. Chichester: Blackwell Publishing Ltd (2007). p. 461– 503. doi: 10.1002/9781118567166.ch25 19. Bernaerts TMM, Verstreken H, Dejonghe C, Gheysen L, Foubert I, Grauwet T, et al. Cell disruption of Nannochloropsis sp. improves in vitro bioaccessibility of carotenoids and ω3-LC-PUFA. J Func Foods. (2020) 65:103770. doi: 10.1016/j.jﬀ.2019.103770 40. Ganzaroli JF, Sanchez JL, Da Silva MV, Tanamati AAC, Fuchs RHB, Tanamati A. Absolute quantiﬁcation of fatty acids in chia seeds produced in Brazil. Bol Cent Pesqui Process Aliment. (2017) 35. doi: 10.5380/cep.v35i1.55932 20. Carbonell-Capella JM, Buniowska M, Barba FJ, Esteve MJ, Frígola A. Analytical methods for determining bioavailability and bioaccessibility of bioactive compounds from fruits and vegetables: a review. Compr Rev Food Sci Food Saf. (2014) 13:155–71. doi: 10.1111/1541-4337.12049 41. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnut.2020. 565996/full#supplementary-material In general, the incomplete bioaccessibility of fatty acids, and to a lesser extent of proteins, is an indication for the expected poor September 2020 \| Volume 7 \| Article 565996 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. REFERENCES Kiczorowska B, Samoli´nska W, Andrejko D, Kiczorowski P, Antoszkiewicz Z, Zajac M, et al. Comparative analysis of selected bioactive components (fatty acids, tocopherols, xanthophyll, lycopene, phenols) and basic nutrients in September 2020 \| Volume 7 \| Article 565996 Frontiers in Nutrition \| www.frontiersin.org 8 Biochemical and Nutritional Evaluation of Microalgae Biomasses Canelli et al. raw and thermally processed camelina, sunﬂower, and ﬂax seeds (Camelina sativa L. Crantz, Helianthus L., and Linum L.) J Food Sci Technol. (2019) 56:4296–310. doi: 10.1007/s13197-019-03899-z 49. Saﬁ C, Frances C, Ursu AV, Laroche C, Pouzet C, Vaca-Garcia C, et al. Understanding the eﬀect of cell disruption methods on the diﬀusion of Chlorella vulgaris proteins and pigments in the aqueous phase. Algal Res. (2015) 8:61–8. doi: 10.1016/j.algal.2015. 01.002 42. Fernandes CE, Vasconcelos MADS, De Almeida Ribeiro M, Sarubbo LA, Andrade SAC, Filho ABDM. Nutritional and lipid proﬁles in marine ﬁsh species from Brazil. Food Chem. (2014) 160:67–71. doi: 10.1016/j.foodchem.2014.03.055 50. Berliner MD. Proteins in chlorella vulgaris. Microbios. (1986) 46:199–203. 51. Capuano E, Pellegrini N, Ntone E, Nikiforidis CV. In vitro lipid digestion in raw and roasted hazelnut particles and oil bodies. Food Funct. (2018) 9:2508–16. doi: 10.1039/C8FO0 0389K 43. Suara SB, Siassi F, Saaka M, Foroshani AR, Asadi S, Sotoudeh G. Dietary fat quantity and quality in relation to general and abdominal obesity in women: a cross-sectional study from Ghana. Lipids Health Dis. (2020) 19:1– 13. doi: 10.1186/s12944-020-01227-5 52. Zhang C, Tang X, Yang X. Overcoming the cell wall recalcitrance of heterotrophic Chlorella to promote the eﬃciency of lipid extraction. J Clean Prod. (2018) 198:1224–31. doi: 10.1016/j.jclepro.2018. 07.114 44. Barbieiri P, Nunes JC, Torres AG, Nishimura RY, Zuccolotto DCC, Crivellenti LC, et al. Indices of dietary fat quality during midpregnancy is associated with gestational diabetes. Nutrition. (2016) 32:656–61. doi: 10.1016/j.nut.2015.12.002 Frontiers in Nutrition \| www.frontiersin.org September 2020 \| Volume 7 \| Article 565996 Conﬂict of Interest: CB and FD were employed by the company Nestlé S.A. Conﬂict of Interest: CB and FD were employed by the company Nestlé S.A. Conﬂict of Interest: CB and FD were employed by the company Nestlé S.A. 45. Attia YA, Al-Harthi MA, Korish MA, Shiboob MM. Fatty acid and cholesterol proﬁles and hypocholesterolemic, atherogenic, and thrombogenic indices of table eggs in the retail market. Lipids Health Dis. (2015) 14:1– 8. doi: 10.1186/s12944-015-0133-z The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 46. Hedenskog G, Enebo L, Vendlová J, Prokeš B. Investigation of some methods for increasing the digestibility in vitro of microalgae. Biotechnol Bioeng. (1969) 11:37–51. doi: 10.1002/bit.260110104 Copyright © 2020 Canelli, Tarnutzer, Carpine, Neutsch, Bolten, Dionisi and Mathys. 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. 47. Morris HJ, Almarales A, Carrillo O, Bermúdez RC. Utilisation of Chlorella vulgaris cell biomass for the production of enzymatic protein hydrolysates. Bioresour Technol. (2008) 99:7723–9. doi: 10.1016/j.biortech.2008.01.080 48. Gille A, Trautmann A, Posten C, Briviba K. Bioaccessibility of carotenoids from Chlorella vulgaris and Chlamydomonas reinhardtii. Int J Food Sci Nutr. (2016) 67:507–13. doi: 10.1080/09637486.2016.1181158 September 2020 \| Volume 7 \| Article 565996 Frontiers in Nutrition \| www.frontiersin.org 9
https://openalex.org/W3115779253	https://revije.ff.uni-lj.si/arshumanitas/article/download/9777/9292	Slovene	null	Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma	Ars & humanitas	2,020	cc-by-sa	13,108	ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Tine Germ 1 Med novejšimi raziskavami Dizmove kronike in zlasti ikonografije emblemov glej Germ 2011 in 2017. 2 Alciati, 1531, A2r-A2v. 1 Uvod Spominska knjiga ljubljanske plemiške družbe sv. Dizma ali krajše Dizmova kronika, hranjena v Arhivu Republike Slovenije (AS 1073, I/1), je bila v zadnjih dveh desetle tjih deležna pozornosti predvsem zaradi edinstvenih, izredno zanimivih emblemov. Ljubljanski rokopis, ki so ga zasnovali in soustvarjali člani leta 1688 ustanovljene plemiške družbe sv. Dizma, ni klasična emblemska knjiga, temveč izvirna oblika akademske spominske knjige, v kateri so predstavljeni emblemi, imena njenih čla nov, njihovi grbi in podatki o vpisu v družbo. Prve miniature so nastale leta 1689, za dnji člani pa so bili vpisani leta 1801.1 Kakovost iluminacij in njihovo ikonografsko bogastvo sta izjemna, tako da Dizmova kronika upravičeno velja za najdragocenejši baročni iluminirani rokopis, kar se jih je ohranilo na Slovenskem. Med emblemi so z ikonografskega vidika še posebej zanimivi tisti, ki vključujejo živali kot nosilce alegoričnega sporočila. Število tovrstnih emblemov in pester nabor živali jasno od ražata zanimanje naročnikov za živalsko simboliko kot tudi široke možnosti izraža nja emblemskega sporočila, ki jih ponuja. Hkrati lahko v tem prepoznamo odmev sočasnih evropskih tokov na področju emblematike, v kateri imajo živali od vsega začetka pomembno vlogo. Priljubljenost živalskih motivov v Dizmovi kroniki je po gojena tudi z dejstvom, da gre za spominsko knjigo, v kateri so natančno naslikani grbi vseh članov. Grbi v večini primerov vključujejo heraldične živali, kar je vplivalo tudi na izbiro živalskih protagonistov v emblemih naročnikov. Prominentna vloga živali v evropski heraldiki je pomembno zaznamovala že samo genezo renesančnega emblema. (Spomnimo, da Alciati, avtor prve emblemske knjige, uvodni emblem v znameniti Emblematum liber zasnuje na podobi grbovnega ščita mi lanskih vojvod in v njem razlaga pomen kače s človeško figuro v ustih.2) Vpliv heraldi ke na emblematiko ostaja stalnica v razvoju emblemskih knjig, neposredna povezanost pa je prav posebej očitna v primerkih, ki so posvečeni vladarjem, visokemu plemstvu, DOI:10.4312/ars.14.2.181-210 181 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma cerkvenim dostojanstvenikom in drugim odličnikom,3 ter v posameznih emblemsko zasnovanih knjigah renesančnih akademij in spominskih knjigah plemiške mladine, ki je študirala na različnih evropskih univerzah (t. i. alba amicorum ali libri amicorum). 4 Alba amicorum imajo z Dizmovo kroniko vsaj dve bistveni skupni lastnosti: nastajali so kontinuirano, v daljšem obdobju kot odprti tip ilustrirane knjige ali vezanih rokopisnih listov z bolj ali manj sofisticiranimi iluminacijami, ob tem pa imajo njihovi vpisni listi neredko podobno strukturo kot strani v ljubljanski Spominski knjigi. Vizualno izpostavljena grb in emblem, dopolnjena z osebnimi podatki in datumom vpisa, se skupaj pojavljata znotraj istega vpisa ter oblikujeta zaokroženo likovno in vsebinsko celoto. 5 Plemiška družba sv. Dizma je uporabljala tudi latinski naziv Academia Unitorum, torej Akademija Zedinjenih, člani pa so se imenovali Academici Uniti. 6 Navajanje folijev v Dizmovi kroniki sledi foliaciji, ki jo je za faksimilno izdajo leta 2001 pripravila N. Golob. 7 Dolničar je kot prvi tajnik Dizmove družbe in njen idejni vodja izdelal tudi nekaj študijskih skic emblemov za člane družbe. Njegova Konceptna knjiga (Semeniška knjižnica, Ljubljana, sign. Rokopis 6) vsebuje 83 skic za emblemske sličice v ovalnih kartušah, eno kartušo, v kateri je samo osnutek srca, in kar nekaj praznih kartuš (Baraga, 2001, 235). 3 Med prvimi tovrstnimi knjigami glej: Giovanni Battista Pittoni, Imprese di diversi prencipi, duchi, signori, e d’altri personaggi et huomini letterati et illustri …, Benetke 1562; Girolamo Ruscelli, Imprese illustri …, Benetke (vol. I–III. 1572, Libro quarto 1583). Alba amicorum imajo z Dizmovo kroniko vsaj dve bistveni skupni lastnosti: nastajali so kontinuirano, v daljšem obdobju kot odprti tip ilustrirane knjige ali vezanih rokopisnih listov z bolj ali manj sofisticiranimi iluminacijami, ob tem pa imajo njihovi vpisni listi neredko podobno strukturo kot strani v ljubljanski Spominski knjigi. Vizualno izpostavljena grb in emblem, dopolnjena z osebnimi podatki in datumom vpisa, se skupaj pojavljata znotraj istega vpisa ter oblikujeta zaokroženo likovno in vsebinsko celoto. Pl išk d žb Di j blj l t di l ti ki i A d i U it t j Ak d ij 1 Uvod Ob tem velja spomniti, da so poleg akademskih emblemskih knjig prav alba amicorum eden od pomembnih zgledov, ob katerih so se navdihovali člani Dizmove družbe.4 Vloga heraldičnih živali pri oblikovanju emblemov članov Akademije Zedinjenih5 se odraža tudi v njihovem izboru: najpogostejši je namreč orel, žival, ki si v evropski heraldiki skupaj z levom deli prvo mesto tako z vidika pogostosti pojavljanja kot z vi dika odličnosti in bogastva simbolnih pomenov. Ko govorimo o priljubljenosti orla v emblemih imenitnih Kranjcev, združenih v družbi sv. Dizma, ne smemo pozabiti, da je orel heraldična žival Vojvodine Kranjske in da so ga zaradi tega včasih v embleme vključili tudi plemiči, ki kraljevske ptice niso imeli v svojem grbu. O pomenu kranjskega grbovnega orla v ljubljanski Spominski knjigi posebej nazorno priča vpisna stran grofa Janeza Henrika Watzenberga (fol. 63r).6 Ta namreč nima klasične emblemske strukture, ampak se napisni trak z devizo In magnis voluisse sat est (V velikih rečeh sama je volja dovolj) ovija kar okrog vratu mogočnega črnega orla, ki razširjenih peruti stoji na vrhu naročnikovega grba. Dva enako velika orla sta upodobljena tudi ob grbovnem ščitu, v katerem nastopa še dvoglavi cesarski orel habsburške monarhije. Orel nad ščitom se s svojo držo in veličastjem nedvoumno spogleduje s heraldično ptico dežele Kranjske, po drugi strani pa s tem, ko nosi napisni trak z geslom, aludira na naročnika. Takšno razlago potrjuje primerjava s študijsko skico, ki jo je za Watzenbergov emblem pripravil Janez Gregor Dolničar v svoji Konceptni knjigi (sl. št. 27).7 Na njej je orel upodobljen v kartuši, na prsih pa ima vrisano stilizirano srce, simbol plemiške družbe sv. Dizma. Andrej Trost (1643–1708), vodilni iluminator v zgodnjem obdobju nastajanja ljubljanske Spominske knjige in verjetni avtor miniature, se je odločil, da ne bo dosledno sledil predlagane mu Dolničarjevemu konceptu in je v končni obliki Watzenbergovega emblema naslikal orla brez stiliziranega srca na prsih. Posledično je simbolna vez z Dizmovo družbo manj očitna, identifikacija s kranjskim heraldičnim orlom pa še bolj izpostavljena in potrjuje 182 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma vlogo heraldike v emblemih kranjskih akademikov. Hkrati gre za prvi primer aktivnega posega slikarja v samo ikonografsko zasnovo emblema, ki razkriva pomemben vidik ge neze emblemov v Dizmovi kroniki, s katerim se bomo še srečali. V Spominski knjigi se orel kot nosilec emblemskega sporočila največkrat pojavlja v povezavi s soncem. 1 Uvod Motiv orla in sonca je zaradi uveljavljene ikonografske tradicije v em blematiki zelo priljubljen, vendar pogostost in raznolikost motiva v emblemih kranjskih akademikov vzbujata pozornost in kličeta po razlagi. V Dizmovi kroniki se namreč poja vi kar desetkrat in vsakič ima specifično pomensko noto. V ikonografskem katalogu, ki je nastal ob faksimilni izdaji Spominske knjige ljubljanske plemiške družbe sv. Dizma, Jože Kastelic (2001, 115–261) ne ponudi ikonografske razlage za omenjene embleme. Prav tako se z motivom ni ukvarjal nihče od starejših avtorjev, ki so pisali o knjigi ljubljanskih akademikov. Pričujoča raziskava je prvi poskus ikonološke analize izbranih emblemov v primerjalnem okviru evropske emblematike. Kasteličev katalog s predikonografskim opisom in prevodom besedilnih delov (akademsko ime člana, izbrano geslo ter dodatni napisi)8 predstavlja dragoceno izhodišče za nadaljnje raziskave, ki, razumljivo, zahte vajo vpogled v ikonografijo orla v evropski kulturi od antike do baroka ter poznavanje emblematike 16. in 17. stoletja. Številni tvorci emblemskih knjig ob razlagi emblemov v potrditev svojih besed navajajo odlomke iz del klasičnih avtorjev, cerkvenih očetov, srednjeveških piscev in svojih sodobnikov, s čimer gradijo hermenevtični korpus, ki ima za raziskovalce neprecenljivo vrednost. Posebno mesto v tem pogledu pripada Filippu Picinelliju, ki v obsežni enciklopediji emblemov Mundus Symbolicus predstavi več kot sto emblemov, v katerih je orel glavni nosilec alegoričnega sporočila (I, 4, embl. 89–217).9 Z vidika pričujoče študije je Picinellijeva zbirka emblemov toliko pomembnejša, ker je bila zelo popularna tudi med kranjskimi intelektualci in predstavlja enega od poglavitnih virov za embleme v Dizmovi kroniki (Germ, 2017, 151–152). 8 Prevod latinskih delov besedila je oskrbel K. Gantar. Avtor članka jih z redkimi izjemami navaja dobesedno. V primeru manjših odmikov od Gantarjevega prevoda je na to dosledno opozorjeno. 9 Picinellijeva knjiga je prvič izšla v italijanskem jeziku Mondo simbolico … v Milanu leta 1653. Kmalu je bila prevedena v latinščino in večkrat ponatisnjena: Mundus symbolicus: in emblematum universitate formatus, explicatus ... justo volumine auctus et Latinum traductus a R. D. Augustino Erath, Köln 1681. Vsi navedki Picinellija se nanašajo na kölnsko izdajo iz leta 1681, zato je uporabljena skrajšana oblika z navedbo zvezka (rimske številke) ter številko knjige in emblema (arabske številke). 8 Prevod latinskih delov besedila je oskrbel K. Gantar. Avtor članka jih z redkimi izjemami nava dobesedno. V primeru manjših odmikov od Gantarjevega prevoda je na to dosledno opozorjeno. 10 Navedene letnice se nanašajo na leto vpisa v Dizmovo kroniko. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Pogostost motiva orla in sonca v emblematiki ne preseneča, saj v evropski kulturi kon tinuirano živi vse od antike, v zgodnjem krščanstvu in srednjem veku pa doživlja in tenziven razvoj zlasti na področju sakralne ikonografije. Zlivanje antičnih tem s kr ščanskimi daje izhodišče za večino motivov, ki jih najdemo v renesančni emblematiki. Hkrati se prav v tem okviru razvijejo tudi nove vsebine, ki jih v starejših obdobjih ne srečamo. V emblemih Dizmove kronike prevladuje tematski sklop, ki se navezuje 183 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma na kristološko ikonografijo orla in sonca, z njim pa se povezujejo alegorične vsebine preroda duše, odrešenja in večnega življenja ter predstave o kontemplaciji, sledenju Kristusovemu nauku in duhu, ki se dviga v nebeške višave. Posebej so izpostavljeni štirje motivi, ki izvirajo iz antike, a so v krščanstvu dobili nove, precej bolj kompleksne vsebine: orel, ki nemoteno zre v sonce; orel, ki leti proti soncu; preizkušnja mladih or ličev z gledanjem v sonce; orel, ki uči mladiče leteti. Ob tem je treba imeti pred očmi dejstvo, da se v emblematiki krščanski koncept spoznavajočega duha, ki se dviga v višave, povezuje s humanističnimi predstavami o odličnosti intelekta, ki ga vodi želja po znanju in spoznanju resnice. Intelektualna nota je v emblemih kranjskih akademi kov večkrat jasno naglašena. Enako velja za zanimanje za antiko, zato elementi antične ikonografije, povezane s solarno simboliko orla in orlom kot Jupitrovo sveto ptico, ne presenečajo. Naštete vsebine in motivi se medsebojno dopolnjujejo, zaradi česar je v kompleksnejših emblemih potrebna previdnost pri razlagi alegoričnega sporočila. Prvi v nizu emblemov z motivom orla in sonca pripada Janezu Jožefu pl. Wal lenspergu (fol. 119r), s katerim začenjamo pregled tudi zato, ker je ikonografsko raz meroma preprost, čeprav je osnovna vsebina tudi tu premišljeno dopolnjena z manj očitnimi simbolnimi pomeni (slika 1). Slika 1: Andrej Trost, Emblem Janeza Jožefa pl. Wallensperga (detajl), 1700, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 119r.10 Slika 1: Andrej Trost, Emblem Janeza Jožefa pl. Wallensperga (detajl), 1700, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 119r.10 Emblemska sličica kaže mogočno kraljevsko ptico, postavljeno v prvi plan gričevnate pokrajine, obsijane s soncem, ki se dviga nad hribe na levi strani miniature. Orel stoji 184 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma na tleh, razpira peruti in gleda naravnost v jutranje sonce. 14 Že Homer piše, da je orel med vsemi pticami Zevsu najljubši (Iliada, XVIII, 245–252; XXIV, 315– 321). Tudi Pindar ga imenuje Zevsovo ptico in kralja vseh ptic (Pitijske ode I, 6; Istmijske ode VI, 50; Olimpijske ode XIII, 21), Horacij pa eksplicitno zapiše, da je Jupiter orlu podelil oblast nad vsemi pticami (Ode, IV, 4). Orel kot kralj ptic nastopa pri Ezopu, na primer v basni Stržek in orel ter Orel in govnač (Perry, 1965, basen št. 434 in št. 3). Motiv orla kot Zevsove oziroma Jupitrove svete ptice ter njegova povezanost z idejo vladarstva dosežeta vrhunec v Rimu, kjer orel postane cesarski simbol. 16 Navedeno po: Zucker, 2005, 78–82. Vsi sklici in navedbe iz Fiziologa so vzeti iz kritične izdaje grškega teksta (rokopis Fiziologa, ki ga hrani Pierpont Morgan Library, Ms 397) s francoskim prevodom Arnauda Zuckerja (Zucker, Paris 2005). Navajanje sledi uveljavljeni tradiciji: najprej navedba skupine, ki ji rokopis pripada, in nato navedba poglavja. Zaradi preglednosti je v opombi dodana stran pariške izdaje. 15 V posameznih interpretacijah motiva gledanja v sonce oziroma letenja proti soncu je lahko orel simbol Kristusa, sonce pa simbol Boga Očeta. Tako v spisu Psevdo Ambroža beremo: »Aquilam in hoc loco Christum Dominum nostrum debemus accipere, qui post venerandam resurrectionem … velut aquila revolavit ad patrem …« (Sermo 46. De Salomone, 5, PL 17, 718) 11 V katalogu (Kastelic, 2001, 152) je geslo prevedeno kot: »Gleda v svetlobi, ki ne zatemni.« Gantarjev prevod ni najbolj posrečen: ne ujema se z vsebino emblema niti z uveljavljeno ikonografijo sorodnih emblemov v knjigah zgodnjega novega veka. 12 »Explorat Phoebum et non caligante tuetur lumine …« (Laurentius Le Brun, Virgilius Christianus, I. Liber Fastorum, sive Hexaemeron & de opere sex dierum, Paris 1661, 433) Verzi so navedeni tudi v večkrat ponatisnjeni zbirki Giovannija Battista Ganduccija, Descriptiones poeticae ex probatioribus poetis excerptae …, Benetke 1676, 114. 13 Prepričanje, da lahko orel nemoteno gleda v sonce, ni iz trte zvito: orlovo oko ima polprozorno membrano (membrana nicitans), ki po potrebi kot tanka veka prekrije zrklo, ga vlaži in varuje, hkrati pa ptici kljub temu omogoča, da vidi. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Posebnost upodobitve je v tem, da lahko na nasprotni strani vidimo sončni zahod, kar je edini primer v ilumina cijah Dizmove kronike, da se sonce v istem prizoru pojavi dvakrat. Nad kartušo je napi sni trak z geslom »Non caligante tuetur lumine« (Zre v svetlobo, ne da bi ga zaslepila),11 pod njo pa krajši, s članskim imenom Der Scharfsichtige (Ostrovidni). Geslo je najver jetneje vzeto iz verzov francoskega pesnika Laurenta le Bruna, v katerih pravi, da orel gleda sonce, ne da bi ga njegova luč zaslepila.12 Motiv Wallenspergove emblemske sličice ima prastare korenine – že Egipčani (pa tudi Grki in Rimljani) so verjeli, da lahko orel zre naravnost v sonce, ne da bi trenil z očmi ali odvrnil pogled.13 To nenavadno sposobnost omenja že Aristotel v Zgodovi ni živali (IX, 34), in povzemajo ga številni antični avtorji, med katerimi je za prenos vsebine v srednji vek bistven Plinij Starejši s svojim Naravoslovjem (X, 3). Alegorične interpretacije se v antiki praviloma povezujejo z Zevsovim orlom, kraljem ptic, ki leta višje kot ostali ptiči in se lahko najbolj približa soncu. Izpostavljajo predvsem njego vo veličastje, moč, plemenitost in pravičnost.14 V krščanstvu dobi motiv orla in sonca nove vsebine: sonce je skladno z razlagami zgodnjekrščanskih piscev praviloma sim bol Kristusa (Christus sol iustitiae), orel, ki se zazira vanj, pa predstavlja vernika ali kr ščansko dušo, ki se preraja v luči prave vere in je deležna odrešitve v Kristusu.15 Prerod in duhovna prenova sta osrednja tema zapisa v Fiziologu (I, 6), kjer je osnovna vsebina orla, ki gleda v sončevo svetlobo, obogatena z motivom leta proti soncu in pomladitvi jo s potapljanjem v bistro vodo.16 Krščanski avtorji ob motivu orlove pomladitve nava jajo besede psalmista, ki pravi: »[…] kakor orlu se obnavlja tvoja mladost« (Ps 103, 5). 185 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Orel je z vstajenjsko simboliko povezan tudi v besedilih zgodnjekrščanskih piscev, na primer Psevdo Ambroža, ki ga v že omenjeni pridigi (Sermo 46. De Salomone) izrecno poveže s Kristusom. Sv. Avguštin v razlagi psalmov orla razume tudi kot prispodobo vstajenja vseh verujočih (Enarrationes in Psalmos, 102, 9, PL 37, 1323). Podoba orlove ga zrenja v sonce je priljubljena tudi v srednjeveških alegoričnih interpretacijah – tako v bestiarijih kot v enciklopedičnih zapisih, eksegetskih besedilih in pridigah. 17 Gessner, Historiae animalium (Lib. III, De avium natura, Zürich 1555, pogl. De aquila, 162–189), Aldrovandi, Ornithologiae, hoc est de avibus historiae Libri XII (Lib. I –II, Bologna 1599, 17–234). Zlasti Aldrovandijeva prva knjiga De aquilis in genere (1599, 17–107) ob navajanju naravoslovnih dejstev vsebuje pravo zakladnico ikonografskih vsebin, razdeljenih na poglavja: Historica, Auguria, Mystica, Hieroglyphica, Fabulae, ter posebno poglavje posvečeno emblemom: Emblemata (str. 72–75). Aldrovandi obširno piše tudi o orlu v heraldiki in likovni umetnosti. 18 Motiv orla in sonca vsebujejo emblemi 98–108, 120, 122, 123, 157, 161, 165, 169, 171, 172, 175–177, 184, 191, 200, 210, 211 (MS I, 4, str. 262–276). 2 Orel in sonce v emblemih članov Akademije Zedinjenih 28 Podoben motiv je za svoj emblem izbral grof Marko Ferdinand Lichtenberg (fol. 287r), le da orel ni na tleh, temveč v gnezdu, postavljenem na visoki pečini. Tudi tu dviga glavo proti soncu in razpira peruti, kot da bo ravnokar poletel (slika 2). Dejstvo, da je gnezdo zgrajeno na skali, dodaja emblemu novo vsebino. Prav tako ne smemo spregledati detaj la, da je v sonce vpisan Kristusov monogram IHS z majhnim križem. Geslo Hoc sidere laetor (Pod to zvezdo je moje veselje) namesto običajnega članskega imena spremlja daljša, opisna različica: Der im Licht deines Antlitzes Wandlende (Tisti, ki hodi v svetlobi tvojega obličja). Gre za aluzijo na psalmistove besede: Blagor ljudstvu, ki pozna veselo vzklikanje, Gospod, v svetlobi tvojega obličja bodo hodili (Ps 89, 16). Geslo in akademsko ime sta usklajena z motivom orla, ki gleda v sonce, poistoveteno s Kristusom, obenem pa sta vsebinsko povezana s podobo božjega obličja, omenjenega v psalmu, na katerega se na vezuje naročnikovo akademsko ime. Hkrati je seveda sonce s Kristusovim monogramom tista »zvezda«, pod katero je Lichtenberg našel »svoje veselje«. Podoben motiv je za svoj emblem izbral grof Marko Ferdinand Lichtenberg (fol. 287r), le da orel ni na tleh, temveč v gnezdu, postavljenem na visoki pečini. Tudi tu dviga glavo proti soncu in razpira peruti, kot da bo ravnokar poletel (slika 2). Dejstvo, da je gnezdo zgrajeno na skali, dodaja emblemu novo vsebino. Prav tako ne smemo spregledati detaj la, da je v sonce vpisan Kristusov monogram IHS z majhnim križem. Geslo Hoc sidere laetor (Pod to zvezdo je moje veselje) namesto običajnega članskega imena spremlja daljša, opisna različica: Der im Licht deines Antlitzes Wandlende (Tisti, ki hodi v svetlobi tvojega obličja). Gre za aluzijo na psalmistove besede: Blagor ljudstvu, ki pozna veselo vzklikanje, Gospod, v svetlobi tvojega obličja bodo hodili (Ps 89, 16). Geslo in akademsko ime sta usklajena z motivom orla, ki gleda v sonce, poistoveteno s Kristusom, obenem pa sta vsebinsko povezana s podobo božjega obličja, omenjenega v psalmu, na katerega se na vezuje naročnikovo akademsko ime. Hkrati je seveda sonce s Kristusovim monogramom tista »zvezda«, pod katero je Lichtenberg našel »svoje veselje«. Ob osnovni vsebini preroda v Kristusu igra tokrat pomembno vlogo tudi motiv orla v skalnem gnezdu, ki ima dolgo ikonografsko tradicijo. 19 Odlomek, ki ga navaja tudi Picinelli (I, 4, 265), najdemo v spisu Meditationes liber unus oziroma Liber Medationum (PL 40, 922), pripisanem sv. Avguštinu. Znanstvena kritika spis danes pripisuje anonimnemu avtorju z zasilnim imenom Psevdo Avguštin. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Motiv v zgodnjem novem veku ohranja svojo aktualnost – najdemo ga tudi v nara voslovnih zapisih. Večkrat ga omenjata oba največja renesančna zoologa Conrad Ges sner (1516–1565) v Historiae animalium in Ulisse Aldrovandi (1522–1605) v knjigah o pticah Ornithologiae, hoc est de avibus historiae.17 V emblematiki je orel, ki gleda v sonce oziroma leti proti njemu, zelo pogost in Picinelli v knjigi Mundus Symbolicus predstavi kar osemindvajset emblemov, ki vključujejo ta motiv.18 Poleg že omenjenih vsebin, povezanih z vstajenjem, razlage največkrat govorijo o odprtosti duha, ki spre jema čisto luč Kristusovega nauka, kontemplaciji in predanosti veri. Pogosto je pou darjena povezava z Janezom Evangelistom, ki temelji na eksegetski ikonografiji: orel, ki se z lahkoto dvigne v nebesne višave in zre naravnost v sonce, je simbol vznesenega duha, ki veje iz besed najmlajšega med evangelisti. Wallenspergovo akademsko ime Ostrovidni in izbrano geslo Zre v svetlobo, ne da bi ga zaslepila kažeta, da moramo emblemsko sličico razbirati v povezavi z idejo ostrovidnega zretja svetlobe v smislu prodornosti duha in kontemplacije (kar seve da ne izključuje s tem povezane ikonografije preroda). Moč duha, da vztrajno sledi soncu, je v sličici poudarjena z na videz nenavadnim pojavom: v istem prizoru sta hkrati vzhajajoče in zahajajoče sonce. Avtor emblema na ta način vnese dimenzijo časa: poudariti želi, da lahko orel nemoteno gleda v sonce od jutra do večera. S tem bralca dodatno opozarja, da je treba prizor razumeti v alegoričnem duhu. Glede na premišljene besedne igre in aluzije, ki jih neredko razkrivajo emblemi članov Dizmove družbe, lahko tudi v primeru Janeza Wallensperga s precejšnjo gotovostjo sklepamo, da je v emblemu skrit še namig na njegovo krstno ime: orel, ki gleda v sonce, je tudi v emblematiki povezan z evangelistom Janezom. Simbolična identi fikacija z naročnikom je dodatno naglašena z dejstvom, da je črn orel heraldična ptica družine Wallensperg. Upodobitev orla v emblemu je povsem enaka kot v grbu, celo krona heraldičnega orla se kot miniaturna kronica ponovi na glavi kralja ptic v emblemski sličici. 186 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Slika 2: Simon Tadej Volbenk Grahovar, Emblem grofa Marka Ferdinanda Lichtenberga (detajl), 1740, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 287r. Slika 2: Simon Tadej Volbenk Grahovar, Emblem grofa Marka Ferdinanda chtenberga (detajl), 1740, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Že Gregor Veliki poduhovljene ga človeka, ki se v iskanju resnice in božje bližine umakne v samoto in višave duha, pri merja z orlom, ki gradi svoje gnezdo visoko v skalah (Moralia in Job, 31, XLVII). Enako piše Psevdo Avguštin,19 ki poduhovljenega človeka primerja z orlom v skalnem gnezdu. 187 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Krščanski avtorji pri alegoričnih razlagah orla v visokem gnezdu najpogosteje jemljejo za izhodišče Jobove besede: »Se orel na tvoje povelje vzdiguje visoko, da si napravi gnezdo v višavi?« (Job 39, 27). Varno gnezdo v vršacih obenem simbolno predstavlja krepost in zaupanje v boga, ki najbolje varuje človeka pred sleherno nevarnostjo. Takšne razlage orla, ki gnezdi v gorah in se zazira v sonce, so v emblematiki znane že v 16. stoletju in jih najdemo tudi pri Picinelliju (I, 4, embl. 123 in 124). Skoraj gotovo je naročnik poznal emblem v knjigi nemškega polihistorja in pesnika Wolfganga Helmharda von Hohber ga Lust- und Artzeney-Garten des Königlichen Propheten Davids (Regensburg 1675, fol. 89r),20 ki ima identično geslo in sorodno ikonografijo.21 Povezava s Hohbergovim em blemom je razvidna tudi v tem, da je osrednji citat njegovega epigrama prav tisti verz iz devetinosemdesetega psalma, iz katerega je Lichtenberg izpeljal svoje akademsko ime. Emblem grofa Lichtenberga se najverjetneje navdihuje tako pri Picinelliju kot pri Hohbergu, vendar vsebuje izvirno vsebinsko nadgradnjo – skupaj z duhovitim slikov nim rebusom namreč razkriva identiteto naročnika. Ideja luči (nemško das Licht), pri sotna tako v besedilnem kot v likovnem delu, je dopolnjena z motivom gore (nemško der Berg), ki se dviga v ospredju krajine, in s tem daje očiten namig na rodbinsko ime Lichtenberg. Kot zanimivost, ki dokazuje, da je pri vsebinski zasnovi emblemov v Spominski knjigi lahko tvorno sodeloval tudi avtor ilustracije, velja omeniti podpis slikarja, Simona Tadeja Volbenka Grahovarja, v katerem izrecno pravi, da je miniaturo naslikal in zasnoval.22 V emblemski sličici Franca Antona Oblaka barona Wolkensperga (fol. 229r) prav tako vidimo orla na visoki pečini, le da se je že dvignil iz gnezda in poletel proti soncu (slika 3). Osnovni pomen duhovnega preroda in vere v odrešenje je podoben kot v emblemu grofa Lichtenberga ter v sorodnih emblemih, ki jih najdemo v Picinellije vi knjigi (MS I, 4, embl. 123, 157, 172, 173, 177, 204). 22 »Grahover inv. (invenit) et pinx. (pinxit)«. Grahovar (1710–1774) je na ta način podpisal še petindvajset miniatur v Dizmovi kroniki. Glede na dolgoletno sodelovanje z naročniki emblemov, njegovo mojstrstvo in inventivnost lahko upravičeno sklepamo, da njegov delež pri vsebinski zasnovi ni bil majhen. Polonca Vrhunc mu pripisuje pomembno vlogo v genezi emblemov in verjame, da je dobro poznal svet simbolov (Vrhunc, 1970, 126). 20 Knjiga vsebuje nemški prevod psalmov z notnimi zapisi novih melodij, molitve, botanične ilustracije z alegorizirajočimi verzi ter embleme z epigrami v latinščini in nemščini. Tiskana knjiga ima 150 vstavljenih pergamentnih folijev z bakrorezi Georga Ch. Eimmarta. Emblemi, ki se vsebinsko povezujejo s psalmi, so na vstavljenih pergamentnih folijih zmeraj na prvi strani, medtem ko so na hrbtni strani bakrorezi rastlin. 21 V Hohbergovem emblemu so v gnezdu tudi mladiči, ki skupaj s staršem gledajo proti soncu. To vnaša nov ikonografski poudarek, saj vključuje motiv preizkušnje orličev z gledanjem v sonce. Da je v primeru Hohbergovega emblema prav ta motiv jedro emblemskega sporočila, potrjuje epigram, ki spremlja sličico. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Vendar ima vsebina emblema posebno noto, izraženo v geslu Nunc procul a strepitu, hic in spe et metu (Zdaj proč od hrupa, tu v upanju in strahu). Gre za ponazoritev želje po odmaknjenosti od hrupnega vsakdanjika v bolj umirjeno poduhovljeno življenje, po iskanju bližine boga, ki se mu človek približuje v upanju in strahospoštovanju. Tudi naročnikovo člansko ime Der 188 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Slika 3: Simon Tadej Volbenk Grahovar, Emblem Franca Antona Oblaka barona Wolkensperga (detajl), 1724, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 229r. Slika 3: Simon Tadej Volbenk Grahovar, Emblem Franca Antona Oblaka barona Wolkensperga (detajl), 1724, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 229r. Verfolgte (Zasledovani) namiguje, da se želi umakniti pred »hrupom« sveta in zaživeti bolj mirno življenje. Ideja je likovno podkrepljena z oblaki, ki zakrivajo vrh gore pred pogledom z nižin. Orel, ki vso pozornost namenja soncu, je obenem heraldična žival rodbine Wolkensperg in simbolično predstavlja naročnika. Takšno razlago potrjuje v slikah izražena besedna igra: gora, zavita v oblake, je aluzija na priimek Wolkensperg, v katerem prepoznamo besedi oblak in gora (nemško die Wolke, der Berg). Da ne bi bilo prav nobenega dvoma, je dodan še detajl rdečega srca na orlovih prsih: srce kot simbol plemiške družbe sv. Dizma na simbolni ravni orla izenačuje z baronom Wol kenspergom, ki ponosno opozarja na svoje članstvo. Verfolgte (Zasledovani) namiguje, da se želi umakniti pred »hrupom« sveta in zaživeti bolj mirno življenje. Ideja je likovno podkrepljena z oblaki, ki zakrivajo vrh gore pred pogledom z nižin. Orel, ki vso pozornost namenja soncu, je obenem heraldična žival rodbine Wolkensperg in simbolično predstavlja naročnika. Takšno razlago potrjuje v slikah izražena besedna igra: gora, zavita v oblake, je aluzija na priimek Wolkensperg, v katerem prepoznamo besedi oblak in gora (nemško die Wolke, der Berg). Da ne bi bilo prav nobenega dvoma, je dodan še detajl rdečega srca na orlovih prsih: srce kot simbol plemiške družbe sv. Dizma na simbolni ravni orla izenačuje z baronom Wol kenspergom, ki ponosno opozarja na svoje članstvo. V emblemu Mihaela Jožefa pl. Wallensperga (fol. 234r) je orel, ki se dviguje k soncu, naslikan nad razburkanim morjem, na katerem je jadrnica, ki se ji je uspelo zasidrati in rešiti pred brodolomom (slika 4). 23 Picinelli predstavi več kot sto emblemov z jadrnico (II, 20, embl. 49–151) in še devet, v katerih ima glavno vlogo sidro (II, 20, embl. 1–9). 24 Topos kreposti in razkoraka med duhovno odličnostjo ter minljivostjo posvetnega življenja najdemo v številnih literarnih delih tako krščanskih kot antičnih avtorjev. Glede na to, da je v Lichtenbergovem geslu uporabljena besedna zveza »mortalia despicit arce«, ki jo v enakem kontekstu najdemo tudi pri Klavdijanu, je možno, da je naročnika navdihnil rimski pesnik (Panegiryus dictus Manlio Theodoro consuli XVII, 1–6). 2 Orel in sonce v emblemih članov Akademije Zedinjenih Varno zasidrana ladja je v evropski ume tnosti tradicionalna podoba rešitve iz stiske in enako velja za področje emblematike.23 Sidro, simbol upanja, je v emblemski sličici jasno izrisano in postavljeno v prvi plan, tako da je njegov pomen še posebej naglašen. Tudi v ljubljanski Spominski knjigi se pojavi večkrat – glej embleme J. Ks. Lichtenthurna (fol. 249r), V. K. Kušlana (fol. 277r), F. A. Schmithoffna (fol. 329r) in K. B. Pettenegga (fol. 341r). V Wallenspergovem em blemu je prispodoba z jadrnico povezana z naročnikom tudi v kontekstu heraldike: na barki vihra zastava z družinskim grbom, orel na nebu pa je hkrati grbovna žival 189 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Slika 4: Simon Tadej Volbenk Grahovar, Emblem Mihaela Jožefa pl. Wallensperga (detajl), 1726, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 234r. Slika 4: Simon Tadej Volbenk Grahovar, Emblem Mihaela Jožefa pl. Wallensperga (detajl), 1726, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 234r. družine. Vsebino sličice dopolnjujeta akademsko ime Der Hoffende (Upajoči) in deviza Ad te levavi oculos meos (Svoje oči vzdigujem k tebi, Ps 123, 1). Izbrano geslo, izrečeno v prvi osebi, celo na jezikovni ravni vzpostavlja neposredno vez med orlom in naročni kom. Orla, ki se dviguje k soncu, lahko tudi tu povežemo z idejo odrešitve in preroda v Kristusu, pri čemer je zanimiva primerjava z emblemom orla in sonca v Mundus Sym bolicus, ki ima identično geslo (I, 4, embl. 102). Slednji sicer ne vključuje motiva barke na morju, vendar izrecno govori o zaupanju v boga, ki nas iz stiske vodi v odrešitev. Na miniaturi grofa Jurija Ludvika Lichtenberga (fol. 238r) se sredi morja dvigu je visoka pečina, vrh katere vidimo orla, ki bo vsak hip poletel proti soncu (slika 5). Nekoliko višje, na levi, drugi orel že leti v smeri sonca. Naročnikov motto ob ideji du hovne prenove tokrat posebej poudarja odličnost duha, ki nas vodi v večno življenje, ter razkorak med vrednostjo večnega na eni in minljivega na drugi strani: Ex hac ma gnanimus mortalia despicit arce, tutius aethereas, ut volet inde vias (S te vzpetine, kdor kvišku se vzpne, prezira umrljivo, varnejša vodi od tod pot ga v višine neba).24 Podobno 190 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Dizma Slika 5: Simon Tadej Volbenk Grahovar, Emblem grofa Jurija Ludvika Lichtenberga 1727, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 238r. Slika 5: Simon Tadej Volbenk Grahovar, Emblem grofa Jurija Ludvika Lichtenberga, 1727, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 238r. 191 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma kot v emblemu Franca Antona Wolkensperga je izhodišče treba iskati v Picinellijevi knjigi, kjer kar šest emblemov izraža podobno vsebino (glej zgoraj). Dejstvo, da sta na sliki upodobljena dva orla – eden že leti proti soncu, drugi pa je še v gnezdu in širi krila, da bi poletel –, lahko razumemo v povezavi z ikonografskim motivom orla, ki spodbuja svoje mladiče k letenju. Pri tem nas ne sme zmotiti dejstvo, da je mladič v gnezdu enako velik kot njegov roditelj: tak način upodabljanja je uveljavljen v rene sančni emblematiki in ima osnovo tudi v naravoslovnih opažanjih.25 Motiv orla, ki z zgledom spodbuja svoje mladiče k letenju, korenini v biblijski eksegezi, v razlagi odlomka iz Mojzesove pesmi Izraelovi skali, v katerem je skrb boga za izbrano ljudstvo ponazorjena s podobo orla, ki širi peruti, uči svoje mladiče leteti in jih hkrati varuje: »Kakor orel, ki spodbuja k letu svoje gnezdo,/ kroži nad svojimi mladiči,/ širi svoje peruti, jih prestreza,/ jih nosi na svojih krilih …« (Mz 5, 32, 11) Prispodoba je med biblijskimi eksegeti izjemno priljubljena in jo najdemo že pri cerkvenih očetih.26 Za kulturno okolje ljubljanske intelektualne elite je zanimivo, da jo v eni od svojih pridig razlaga Janez Ludvik Schönleben.27 Motiv je dobro znan tudi v emblematiki: Picinelli predstavi pet emblemov s to vsebino (I, 4, embl. 175, 186, 194, 197, 214); enako število najdemo tudi v zbirki emblemov Jacoba Boscha Sym bolographia (I, embl. 206, 208; II, embl. 260, 447, 615).28 V emblemih, ki jih navajata Picinelli in Bosch, je lahko alegorično sporočilo glede na geslo in kontekst različno: v okviru sakralne ikonografije gre za ponazoritev idej spodbujanja k življenju v veri in kreposti, dajanja dobrega zgleda, duhovnega vzpona in skrbi boga za krščansko občestvo, v profani pa isti motiv največkrat predstavlja skrb dobrega vladarja za svoj rod, ljudstvo in deželo, ki ji vlada. 25 V obdobju, ko mladi orli vzletijo, so že skoraj odrasli in njihova silhueta na nebu se ne razlikuje bistveno od odrasle ptice. 26 Gl. npr. sv. Ambrož (Hexaemeron, PL 14, 232) ali sv. Hieronim (Breviarium in Psalmos, PL 26, 1097). 27 Joannis Ludovici Schönleben …, Feyertäglicher Erquick-Stunden: Das ist: Ehren- vnd Lobpredigen der lieben Heiligen Gottes… Anderer Theil, Salzburg 1670, 11. 28 Jacob Bosch, Symbolographia sive De arte symbolica sermones septem …, J. C. Bencard, Augsburg/ Dilingen 1702. 30 Picinelli se pri tem sklicuje na Tomaža Akvinskega in njegovo misel, da smo ljudje podvrženi dobremu in slabemu ter da nas božja ljubezen (caritas) vodi k zmagi dobrega. Misel podkrepi z besedami sv. Avguština, ki govori o ognju božje ljubezni, v kateri se dobro razloči od zlega. S tem daje neposredno izhodišče tako za povezavo božje ljubezni z ognjem kakor tudi za motiv preizkušnje in izbire med dobrim in zlim, kar je osnovna vsebina emblema. 29 V krščanski teologiji in ikonografiji je caritas izjemno kompleksen pojem, ki po eni strani predstavlja vseobsežno božjo ljubezen, milost in velikodušnost, po drugi pa ljubezen vernikov do boga in ljubezen do bližnjega (dilectionem Dei et proximi). 29 V krščanski teologiji in ikonografiji je caritas izjemno kompleksen pojem, ki po eni strani predstavlja vseobsežno božjo ljubezen, milost in velikodušnost, po drugi pa ljubezen vernikov do boga in ljubezen do bližnjega (dilectionem Dei et proximi). 30 Picinelli se pri tem sklicuje na Tomaža Akvinskega in njegovo misel, da smo ljudje podvrženi dobremu in slabemu ter da nas božja ljubezen (caritas) vodi k zmagi dobrega. Misel podkrepi z besedami sv. Avguština, ki govori o ognju božje ljubezni, v kateri se dobro razloči od zlega. S tem daje neposredno izhodišče tako za povezavo božje ljubezni z ognjem kakor tudi za motiv preizkušnje in izbire med dobrim in zlim, kar je osnovna vsebina emblema. 29 V krščanski teologiji in ikonografiji je caritas izjemno kompleksen pojem, ki po eni strani predstavlja vseobsežno božjo ljubezen, milost in velikodušnost, po drugi pa ljubezen vernikov do boga in ljubezen do bližnjega (dilectionem Dei et proximi). 30 Picinelli se pri tem sklicuje na Tomaža Akvinskega in njegovo misel, da smo ljudje podvrženi dobremu in slabemu ter da nas božja ljubezen (caritas) vodi k zmagi dobrega. Misel podkrepi z besedami sv. Avguština ki govori o ognju božje ljubezni v kateri se dobro razloči od zlega S tem daje neposredno 31 Glej: Bosch, 1702, II, embl. 1014. 32 Geslo je zaključek verza iz Ovidijevih Metamorfoz: »Inde genus durus summus experiensque laborum / et documenta damus, quia simius origine nati« (I, 414–415). Verzi so navedeni v prevodu Barbare Šega Čeh: Publij Ovidij Nazon, Metamorfoze I.–III., Ljubljana 2013. Gantar verz prevaja nekoliko drugače: »Sami smo živ dokaz tvari, ki iz nje smo rojeni.« (Kastelic, 2001, 204) 33 Zaradi vsebinske povezanosti obeh motivov je njuno stapljanje razumljivo in se uveljavi že v srednjem veku. Kljub temu da antični viri preizkušnjo vida postavljajo v čas, ko so mladiči še puhasti in ne morejo leteti, krščanska ikonografija ta detajl pogosto prezre oziroma se raje opre na poznejše zapise v posameznih bestiarijih, kjer beremo, da orel preizkušnjo vida pri mladičih opravi šele, ko lahko letijo. Skupaj z njimi poleti proti soncu: tiste, ki mu sledijo in zmorejo gledati v sonce, prizna za svoje, ostale zavrže. (Glej poglavje o orlu v bestiariju Guillauma le Clerca, Bestiaire divin, sr. 13. st., Bodleian Library, MS. Douce 132.) 34 Pi i lli d t i d t bl t bi (I 4 bl 89 97) 2 Orel in sonce v emblemih članov Akademije Zedinjenih Orel, grbovna žival grofov Lichtenbergov, skladno z uveljavljeno tradicijo v lju bljanski Spominski knjigi aludira na naročnika, osnovni motiv poti kreposti, ki je v evropski ikonografiji prisoten v najrazličnejših inačicah, pa ima v tem primeru izra ženo dodatno vsebinsko noto, ki izvira iz klasične antike. Ne smemo namreč prezreti dejstva, da se emblemska sličica, ki je sicer še ujeta v kartušo, razpira in se približa odprtemu emblemu. Na mestu, kjer okvir kartuše ostaja odprt, vanjo diskretno vsto pata grofov grb ter lik malega Herakleja z gorjačo in levjo kožo. Ob Herakleju stoji ženska figura z ognjenimi zublji na prsih, ki z desnico kaže nanje, z levico pa mladega junaka opozarja na orla. Čeprav žena ni označena z napisom, lahko glede na kon tekst sklepamo, da ponazarja krepost: bodisi vrlino v širšem pomenu (virtus) bodisi 192 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma najpomembnejšo med krščanskimi krepostmi, to je ljubezen (caritas),29 ki je v moralni alegoriki neredko predstavljena kot lepa mladenka s plameni na prsih. Lichtenbergovo člansko ime Der Grossmütige (Velikodušni) kaže, da ženska figura v resnici pooseblja caritas. Dodatno potrditev lahko najdemo tudi v emblematiki: Picinelli v enem od emblemov motiv orla, ki spodbuja mladiče, da gledajo v sonce, poveže s podobo božje ljubezni (caritas) ter motivom razločevanja med dobrim in slabim (I, 4, embl. 93).30 Lichtenbergov emblem torej vključuje motiv Herakleja na razpotju, ki izbira med po tjo kreposti na eni ter lahkotnim življenjem in zasledovanjem minljivih užitkov na drugi strani. Znani antični motiv, ki je priljubljen tudi v krščanski alegoriki zgodnjega novega veka (Panofsky, 1930, 37–173), smemo razumeti kot neposredno dopolnilo ikonografskega sporočila emblema tudi zato, ker se trak z geslom izjemoma vije na spodnjem robu sličice in se začenja tik nad Heraklejevo glavo. Orla, ki mladiče spodbuja k letenju, si je za svojo emblemsko sličico izbral tudi baron Janez Benjamin Erberg (fol. 264r). V njej vidimo odraslega ptiča, ki je poletel z visoke skale na nabrežju morja in se dviga proti soncu, štirje mladiči pa sledijo njego vemu zgledu (slika 6). Slika 6: Simon Tadej Volbenk Grahovar, Emblem barona Janeza Benjamina Erberga (detajl), 1736, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 264r. Slika 6: Simon Tadej Volbenk Grahovar, Emblem barona Janeza Benjamina Erberga (detajl), 1736, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 264r. 34 Picinelli predstavi devet emblemov s to vsebino (I, 4, embl. 89–97). 31 Glej: Bosch, 1702, II, embl. 1014. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Motiv je pogost tudi v emblematiki, kjer osnovna vsebina preizkušnje dobi številne nove simbolne razlage.34 194 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Vpisni list barona Franca Rudolfa Wolkensperga (fol. 369r) krasi celostranski od prti emblem s pogledom na gorsko krajino, ovito v meglice, skozi katere v nebo štrlijo le najvišji vršaci (slika 7). Na enem od njih je gnezdo z orličem, nekoliko bližje gledal cu pa leti odrasel orel, ki se oddaljuje od gnezda. V obroču oblakov je na nebu sonce z vrisanim božjim očesom, s čimer je naglašena njegova krščanska simbolika. Nad mladičem, ki razpira peruti in dviguje glavo k sončevi luči, je v ravni črti proti soncu izpisano geslo: Auxilium de coelo (Pomoč z neba). V prvem planu slike sta ob naročni kovem grbu in kartuši s podatki o vpisu naslikani poosebitvi pravičnosti in slave. Slava v rokah drži širok bel trak z baronovim članskim imenom: Der Verlassene und doch Beglückte (Zapuščeni, pa vendar osrečeni). Slika, predvsem pa geslo in akademsko ime kažejo, da gre za izvirno različico mo tiva mladega orla, ki se uči leteti. Na Grahovarjevi miniaturi je starš že zletel iz gnezda in mladiču daje zgled, vendar mu orlič ne sledi, kot bi pričakovali glede na ustaljen tip likovnih realizacij v emblematiki.35 »Zapuščen« v gnezdu izteguje glavo proti soncu, ki simbolizira Kristusa, kakor da v svoji šibkosti prosi za pomoč z neba. To slikar v likov nem in vsebinskem pogledu poudari z dvema elementoma: starš se oddaljuje od gnez da, s čimer stopnjuje dramsko napetost, na drugi strani pa z neobičajnim načinom izpisa gesla umetnik vzpostavi vizualno vez med mladičem in soncem. Bralec zlahka razume, da bo mladič, ki ni zbral poguma, da bi sledil staršu, vsak trenutek deležen pomoči: bog, v katerega zaupa, mu bo vlil srčnosti, da poleti. Ob tem velja izpostaviti, da Janez Ludvik Schönleben v že omenjeni razlagi prispodobe o orlu, ki uči mladiče leteti, izrecno poudarja »pomoč z neba«, božjo ljubezen in milost, ki duhu pomagata, da se dvigne kvišku. Govori tudi o opogumljanju in spodbujanju, pri čemer navaja odlomke iz pridige sv. Bernarda (Pridiga o Kristusovem vnebohodu) in pridige na isto temo, ki jo je napisal njegov učenec opat Guerric d'Igny.36 V emblemu barona Wolken sperga je poudarek prav na zaupanju v boga, ki ljubeče skrbi za vse svoje »otroke«, jim vliva pogum in kaže pot. g 36 Schönleben večkrat uporabi priljubljen obrazec »sursum corda«, ki redno nastopa v krščanski liturgiji (1670, 11). Krščanski pisci besedno zvezo najpogosteje uporabljajo v pridigah, v kontekstu opogumljanja vernikov in spodbujanja srčnosti. 2 Orel in sonce v emblemih članov Akademije Zedinjenih 193 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Tokrat so mladiči, v nasprotju z realističnim pristopom, upodobljeni opazno manjši od svojega očeta. Vendar se z morebitnimi vsebinskimi implikacijami ne kaže prenagliti: deloma lahko razliko v velikosti pripišemo kompozicijski zasnovi miniature, saj orliči vzletajo s skale v daljavi in sledijo staršu, ki se približuje prednjemu planu slike, zato je razumljivo večji. Ob tem je treba upoštevati še ikonografsko tradicijo renesančnih emblemov, v katerih so mladiči zaradi lažjega razumevanja motiva neredko namenoma upodobljeni manjši od staršev.31 Baronovo izbrano člansko ime Der Nachartende (Posne majoči) se nanaša na mladiče, geslo Et documenta damus, quia simius origine nati (Sami smo živi dokaz korenin, ki iz njih smo pognali)32 pa vsebino emblema zaokroži z mislijo, da mladiči s tem, ko sledijo zgledu staršev, dokazujejo, da so njihovi vredni nasledniki. Geslo priča, da gre za razmeroma pogosto kombinacijo obeh ikonografskih motivov – spodbude k letenju in preizkušnje mladih orličev, ki jih oče sili, da gledajo v sonce.33 Motiv korenini v antični tradiciji in že Aristotel v Zgodovini živali (IX, 34) piše, da naj bi orel svoje mladiče silil gledati v sonce. Če tega ne zmorejo, jih zavrže, saj je njihova šibkost dokaz, da niso njegovi pravi potomci. Nekatere manjše ujede naj bi namreč v orlovo gnezdo poskušale podtakniti svoja jajca in s tem zagotoviti preživetje lastnega zaroda v varstvu kralja ptic. A ker noben ptič, razen orla, ne more nemoteno gledati v sonce, orlovski starši z omenjeno preizkušnjo odkrijejo podtaknjena piščeta. O nenavadni skušnji poleg Aristotela poročajo tudi Plinij v Naravoslovju (X, 10), Aj lijan v Značilnostih živali (II, 26 in IX, 13), Mark Anej Lukan v pesnitvi Farzalija (IX, 902–906) ter drugi antični avtorji. Motiv v krščanskem duhu alegorično razlaga že sv. Ambrož (natančneje Psevdo Ambrož) v pridigi o Salomonu (Sermo 46. De Salomone, PL 17, 718). Med srednjeveškimi enciklopediki pa Izidor Seviljski v Etimologijah (XII, 7, 11), na katerega se sklicujejo poznejši pisci. Neobičajna epizoda je redno predsta vljena tudi v bestiarijih, pri čemer najpogostejša alegorična razlaga pravi, da so orliči, ki gledajo v sonce, podoba vernikov, ki v polni meri sprejemajo božji nauk in so zato deležni preroda. Mladiči, ki tega ne zmorejo, predstavljajo slabe kristjane, grešnike ali nevernike. 35 Kastelic ob opisu dodaja, da orel leti v dolino po plen (2001, 252), čeprav za takšno razlago v emblemu ni nobene ikonografske osnove in tudi sicer v emblematiki nima ustreznih vzporednic. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Poosebitvi v sprednjem delu slike vsebino dopolnjujeta s spo ročilom, da je na poti, ki vodi v večno slavo, pomembna pravičnost in z njo povezana ljubezen do resnice. Pravilnost razumevanja emblemskega sporočila potrjujeta položaj in način upodobitve obeh personifikacij: Pravičnost, ki nas vodi v življenju, se naslanja na baronov grb in se s pogledom osredotoča nanj, Slava pa dviguje svoj pogled v nebo, kamor se pne trak s članskim imenom naročnika. Dejstvo, da ima figura pod nogami zemeljsko oblo, da je bosa in oblečena v preprosto belo obleko, kaže, da ne gre za obi čajno idejo slave, temveč za krščansko poosebitev večne slave. Kot že v primeru Franca Antona Wolkensperga vršaci z oblaki tudi tokrat namigujejo na rodbinsko ime. 195 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Slika 7: Simon Tadej Volbenk Grahovar, Emblem barona Franca Rudolfa Wolkensperga, 1760, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 369r. Slika 7: Simon Tadej Volbenk Grahovar, Emblem barona Franca Rudolfa Wolkensperga, 1760, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 369r. 196 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Slika 8: Bartolomej Ramschissl(?), Emblem Franca Mihaela pl. Erberga (detajl), 1709, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 161r. Slika 8: Bartolomej Ramschissl(?), Emblem Franca Mihaela pl. Erberga (detajl), 1709, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 161r. Motiv orla, ki mladiče spodbuja k letenju, je z nekoliko spremenjenim vsebinskim poudarkom ob svojem vpisu v Dizmovo družbo izbral tudi Franc Mihael pl. Erberg (fol. 161r) (slika 8). V emblemski sličici trije orli letijo proti soncu, medtem ko je četrti še na tleh in razpira peruti, da bi poletel. Ob tem ne moremo spregledati, da tokrat vlogo sonca deloma privzema veliko rdeče srce: naslikano je tik pred son cem, ujeto v njegov svetlobni sij, kot da tudi samo izžareva svetlobo. Živordeče srce, simbol družbe sv. Dizma, lahko opazimo tudi v kljunu orla, ki se pripravlja, da bo vzletel. Na napisnem traku znotraj kartuše je kratko geslo Non degenero (Ne spridim se), na daljšem zunaj nje pa je zapisano akademsko ime Der Ehrbahre (Častivredni). Motto kot osrednjo temo izpostavlja preizkušnjo orličev. Geslo ni izvirno, temveč povzeto po emblemu Karla Emanuela I., vojvode Savojskega, ki govori o tem, da je vojvoda vreden sin svojega očeta (MS I, 4, embl. 97). 2 Orel in sonce v emblemih članov Akademije Zedinjenih Picinellijeva razlaga pokaže, da ne gre samo za identično geslo, temveč je tudi vsebina emblema precej podobna, le da je pri Erbergu dopolnjena z namigom na njegovo članstvo v Dizmovi družbi. Če ne bi motto očitno kazal na preizkušnjo mladičev, bi lahko pomislili, da je glav na tema sličice spodbujanje mladih orlov k letenju. V resnici gre za stapljanje obeh motivov, pri čemer je zaradi gesla in akademskega imena v prvem planu ideja časti vrednega sledenja zgledu prednikov. Izvirna nadgradnja osnovne ikonografije je pri Erbergovem emblemu vidna v tem, da se znak Dizmove družbe simbolično povezu je s soncem, obenem pa enako srce drži tudi orel, ki se pripravlja za vzlet. S tem se ideja emblema preoblikuje v duhu, ki ga v Spominski knjigi večkrat zasledimo: sle denje načelom družbe sv. Dizma in izpolnjevanje skupnih zavez prispevajo k njeni odličnosti, hkrati pa plemenitijo vsakega posameznega člana. Motiv skušnje orlovih 197 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma mladičev je torej domiselno cepljen na temo pripadnosti plemiški družbi in njenim načelom, predvsem pa sledenju zgledov starejših članov. Dejstvo, da so vsi štirje orli videti kot odrasle ptice, je v tem primeru najverjetneje zavestna odločitev, povezana z emblemskim sporočilom. Orel, ki drži v kljunu živorde če srce, simbolno predstavlja naročnika, kar potrjujejo tudi člansko ime, geslo in pove zava s heraldičnim orlom Erbergov. Franc Mihael Erberg – Častivredni – z vključitvijo stiliziranega srca sporoča, da sledi vzoru starejših članov Akademije Zedinjenih. Kako spretno je neznani avtor sličice (verjetno Trostov učenec Bartolomej Ramschissl) na glasil emblemsko sporočilo, postane v celoti vidno ob primerjavi z Dolničarjevo skico, na kateri trije enako veliki orli s srci v kljunih letijo proti večjemu srcu na nebu (Kon ceptna knjiga, sl. št. 75). Slikar je trem orlom dodal četrtega, ki ga je s postavitvijo v ospredje kompozicijsko in velikostno izpostavil, hkrati pa mu je kot edinemu dodelil srce, ki ga dviguje kvišku.37 Slikarjev poseg daje emblemu nov vsebinski poudarek in priča o njegovi aktivni vlogi v sami ikonografski zasnovi. To ni edini primer, ko ume tnik dejavno sooblikuje emblemsko sporočilo – Andrej Trost je svobodno predrugačil Dolničarjevo skico orla za Watzenbergov emblem (glej zgoraj) in zanesljivo preobli koval skicirani koncept vsaj še v primeru emblema za Janeza Štefana Florjančiča (fol. 73r), kjer je poseg z ikonografskega vidika še radikalnejši (Germ, 2017, 156–157). 38 Orla v povezavi z Zevsom in njegovo strelo omenja že Pindar v Pitijski odi (I, 5–10), večkrat pa ga srečamo v rimskem pesništvu, na primer v Ovidijevih Metamorfozah (X, 155), Horacijevih Odah (IV, 4) ali Vergilijevi Eneidi (V, 252). O orlu in streli piše tudi Plinij v Naravoslovju (X, 4, 15). Orla, ki v krempljih drži strelo, najpogosteje najdemo na helenističnih kovancih Ptolemajske dinastije in seveda na rimskih novcih. V Rimu je orel s strelo eden najpomembnejših državnih simbolov. 37 Ob tem je avtor emblemske sličice vnesel še eno spremembo: v Dolničarjevi skici Erbergovega emblema je namesto sonca na nebu narisano shematizirano srce, slikar pa je srce obdal s sončevim sijem ter ga tako likovno kot ikonografsko najtesneje povezal s soncem. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Vse binski poudarek, ki ga je s svojo intervencijo izpostavil miniaturist, pojasni tudi, zakaj orel, ki vzleta, ni upodobljen kot mladič. Osnovna ideja pristnosti rodu in častivredno sti nasledstva (ki prevladuje v emblemu Emanuela Savojskega) se, kot rečeno, poveže s priložnostjo grofovega vpisa v plemiško družbo sv. Dizma in poudarja, da se hoče izkazati vrednega zaupanja akademskih kolegov, ki so ga sprejeli medse. Odprti emblem grofa Volfa Engelberta Ignaca Auersperga (fol. 306r) kaže obmor sko pokrajino z mogočnimi ruševinami, pred katerimi stojijo štirje moški temne polti, ki z loki streljajo na orla. Kralj ptic leti k soncu in je tako visoko na nebu, da ga puščice mož s perjanicami ne morejo doseči. Jalovost njihovega početja potrjuje geslo na napi snem traku v orlovih krempljih: Irrideo tela (Ne zmenim se za strelice). Grofovo člansko ime Der Hochschwebende (Visoko lebdeči) zaokroži podobo orla, ki leta višje kot vse ptice in mu puščice ne morejo do živega (slika 9). Že stari Grki in Rimljani so verjeli, da orel leta tako visoko, da mu niti strele ne morejo škodovati. Nasprotno, ptica, ki spremlja vladarja bogov, lahko celo nosi Zevso ve strele v svojih krempljih.38 Motiv orlovega visokega leta se v krščanski ikonografiji 198 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Slika 9: Simon Tadej Volbenk Grahovar, Emblem grofa Volfa Engelberta Ignaca Auersperga, 1741, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 306r. Slika 9: Simon Tadej Volbenk Grahovar, Emblem grofa Volfa Engelberta Ignaca Auersperga, 1741, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 306r. 199 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma uveljavi predvsem s sv. Avguštinom, ki intelektualno zahtevnost Janezovega evangelija v odnosu do ostalih treh evangelijev primerja z letom orla, s čimer dobi motiv alegorično dimenzijo posebne duhovne moči in odličnosti.39 Obe tradiciji se zlijeta v emblemati ki, kjer je motiv zelo pogost in ima celo paleto simbolnih vsebin. V sonce je – kot pri mnogih emblemih Dizmove kronike – vrisano božje oko. S tem je vsebinsko izhodišče emblema nedvoumno povezano z ikonografijo preroda v Kristusu, vendar ima sporočilo emblema tokrat bolj izpostavljeno idejo vzvišenosti nad nevarnostmi in tegobami tega sveta: kdor stremi k višjim ciljem, najde zavetje v bogu in mu zlo ne pride do živega. 39 Sv. Avguštin, In Evangelium Joannis tractatus, XXXVI, 5, PL 35, 1668. 40 »Indijanci« s peresnimi kronami in kratkimi pisanimi krilci, oboroženi z loki in puščicami, so značilni predstavniki Amerike, poimenovanje pa izvira iz Kolumbovega zmotnega prepričanja, da je odkril Indijo. Vse od 16. stoletja jih najdemo v alegoričnih prizorih (predvsem v okviru ikonografije štirih kontinentov), v ilustriranih knjigah s področja geografije in zgodovine, v potopisnih delih in drugje. 41 »Aut quos Oceano propior gerit India lucos,/ Extremi sinus orbis: ubi aera vincere summum/ Arboris haud ullae jactu potuere sagittae?/ Et gens illa quidem sumits non tarda pharetris« (Georgike II, 122–125). 39 Sv. Avguštin, In Evangelium Joannis tractatus, XXXVI, 5, PL 35, 1668. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Ob tem ostaja odprto vprašanje, zakaj je slika orla, ki ga puščice ne dosežejo, dopol njena z eksotičnim motivom »indijanskih lokostrelcev«.40 V emblemskih knjigah takšne podobe ne najdemo – sličice se omejujejo na poenostavljeno predstavitev orla, ki leti viš je od puščic. Ključ do razumevanja ikonografskega oreha lahko najdemo v Simbolografiji Jacoba Boscha, v emblemu, posvečenem ideji plemenitega državnika, ki je vzvišen nad obrekovanji in spletkami (III, embl. 1025). Bakrorez z orlom in puščicami je namreč po spremljen z geslom Haud ullae potuere (Nikakor ne more), avtorjeva notica ob emblemu pa opozarja, da je motto povzet po Vergiliju. V resnici gre za besede iz verza, ki ga najde mo v Georgikah, ko pesnik ob omembi Indije govori o drevesih, katerih krošnje so tako visoke, da nobena puščica ne more poleteti do njihovega vrha, pa čeprav so domorodci izvrstni lokostrelci.41 Motiv orla, ki ga puščice ne dosežejo, je torej pri Boschu povezan z Indijo in Vergilijevo prispodobo o višini tamkajšnjih dreves, pictura pa je skladno z lapidarnostjo kompozicij, ki je značilna za emblemske sličice v Boschevi enciklopediji, omejena na najnujnejše likovne prvine: orla v letu in puščice pod njim. Grahovar je imel na voljo celo stran in je sporočilo Auerspergovega emblema do polnil s slikovitim prizorom »indijanskih« lokostrelcev, ki so v resnici Indijci, o katerih piše Vergilij. Slikar in naročnik sta lahko likovne zglede za eksotičen motiv »Indijancev« našla v različnih virih – od alegoričnih predstavitev kontinentov, kjer spremljajo poose bitev Amerike, do ilustracij v geografskih atlasih ali zgodovinskih zapisih o Novem svetu. Zagotovo gre pri tem tudi za odmev takrat modnega navdušenja nad eksotičnimi deže lami. Hkrati lahko z veliko verjetnostjo zapišemo, da je posredno prisotna tudi aluzija na antično zanimanje za čudesa Orienta, kot ga je v pesniških podobah naslikal Vergilij. Turški paša, ki sedi ob kartuši in se lagodno naslanja na prevrnjen kamniti blok z vklesanim grbom rodbine Auersperg, ni neposredno vključen v sporočilo emblema, razumemo pa ga lahko kot izraz istega navdušenja nad eksotičnim, ki ga izpričujejo 200 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma »Indijanci« v fantazijski opravi. Slikoviti orientalski motivi z moškimi in ženskimi liki v otomanski noši se pojavljajo tudi v nekaterih drugih emblemih Dizmove kronike, na primer v emblemu barona Marka Antona Bilichgrätza (fol. 320r) ali barona Karla Leopolda Rosettija (fol. 357r). Vsi liki v otomanskih oblačilih se umeščajo v modno tradicijo t. i. 2 Orel in sonce v emblemih članov Akademije Zedinjenih turkerij, ki so v tem času priljubljene v številnih evropskih deželah, seveda tudi na ozemlju današnje Slovenije. V primeru turjaških knezov bi turškega pašo lahko razumeli kot namig na slavno zgodovino naročnikovih prednikov, ki so se borili proti Turkom. Slednji so bili še ne tako dolgo nazaj huda nevarnost za Kranjsko in velik del Srednje Evrope, dokler niso bili uničujoče poraženi v bitki pri Sisku leta 1593. V njej je pomembno vlogo odigral slavni prednik naročnika Andrej Auersperg (1557–1593), ki se je s svojim junaštvom zapisal v zgodovino. Kranjska poslej ni bila več tako neposre dno ogrožena, saj je otomanski imperij ofenzivo preusmeril bolj na sever proti cesarski prestolnici na Dunaju. Posledično so se v slikarstvu Turki iz krvoločnih vojščakov po gosto prelevili v eksotične like, ki gledalce razveseljujejo s svojo slikovito nošo. V nizu emblemov z motivom orla in sonca v ljubljanski Spominski knjigi pripada emblemu barona Karla Jožefa Valvasorja (fol. 294r) posebno mesto: je namreč eden od redkih, ki ima ob geslu in članskem imenu še dodatno pojasnilo v smislu kratke ga epigrama (slika 10). Izbrano akademsko ime Der Verneuerte (Prenovljeni) in geslo Formosus renascens (Lepši ob ponovnem rojstvu) tako dopolnjujeta verza: Ex senio re diturus adit Iovi armiger aetheram / Solis ubi hanc puro sidere flamma cremat (Jupitrov orel se vrača iz starosti in k nebu se vzpenja, tja, kjer sončni ga plamen s prečistim sijajem Slika 10: Simon Tadej Volbenk Grahovar, Emblem barona Karla Jožefa Valvasorja (detajl), 1740, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 294r. Slika 10: Simon Tadej Volbenk Grahovar, Emblem barona Karla Jožefa Valvasorja (detajl), 1740, Dizmova kronika, ARS, Ljubljana, AS 1073, I/1, fol. 294r. 201 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma objame). Motiv preroda je izrecno izpostavljen; v ikonografskem pogledu je sporočilo emblema tako jasno, da o njem ne bi bilo treba izgubljati besed, če ne bi v obstoje či ikonografski interpretaciji prišlo do nepričakovanega zdrsa. Avtor ikonografskega kataloga namreč opisu emblemske sličice doda kratko obrazložitev: »Slika orla v an titetični obliki potrjuje devizo« (Kastelic, 2001, 217). Očitno je njegovo pozornost pri tegnilo dejstvo, da orel v nasprotju z ostalimi emblemi v Dizmovi kroniki ni naslikan v letu proti soncu, ampak leti proč od njega, pri čemer mu iz peruti izpadajo peresa. 44 Bosch ob sličici orla, ki je obrnjen stran od sonca in mu iz kril odpadajo peresa, ponudi tri gesla: Renovata juventus, Et damna juvabunt in Donec renover (Bosch I, embl. 853). Izrecno utemeljitev za motiv orla, ki mu stara peresa izpadejo potem, ko se že oddaljuje od sonca, najdemo med drugim v priljubljeni enciklopediji Bartolomeja Angleža O lastnostih stvari (ok. 1342–1347), kjer piše, da vročina sonca orlu razširi pore, zaradi česar mu začnejo peresa izpadati (De proprietatibus rerum, XII, 1). 42 Za prepričanje, da se je orel sposoben pomladiti, glej tudi Izaijo: »[…] tisti pa, ki zaupajo v Gospoda obnavljajo svojo moč, / vzdigujejo trup kakor orli« (Iz 40, 31). j j j g j j p 43 Za celotno poglavje o orlu glej: Zucker, 2005, 78. j j j g j j p 43 Za celotno poglavje o orlu glej: Zucker, 2005, 78. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Kasteličeva trditev je problematična že v izhodišču, saj je v neskladju s samo teorijo renesančnega emblema: vsi nosilci sporočila morajo biti organizirani po načelu med sebojnega dopolnjevanja, tako da skupaj izoblikujejo homogeno emblemsko vsebino. Z drugimi besedami, besedilni in slikovni del emblema ne moreta biti v antitetičnem razmerju na način, kot predlaga Kastelic. V emblemu pravzaprav ni nobene antitetičnosti, saj pictura smiselno ilustrira raz širjeno različico motiva orlovega preroda, kakršno ponuja že Fiziolog. Ostareli orel, beremo, poleti k soncu, ki mu sežge staro perje ter razblini meglico v motnih očeh, nato pa se trikrat potopi v bistro vodo, iz katere vzleti popolnoma prerojen. Vero dostojnost trditve avtor Fiziologa podkrepi z besedami psalmista: »[…] kakor orlu se obnavlja tvoja mladost.« (Ps 102, 5),42 ob tem pa dodaja alegorično razlago: »Kadar ti oblačila starega človeka postanejo težka in oči tvojega srca motne, poišči izvir duha, živi vir, ki je Gospodova beseda […] in poleti v višave proti soncu pravice, Jezusu Kristusu ter se osvobodi oblačil starega človeka in njegovega početja« (I, 6).43 Čeprav epigram, v katerem je kralj ptic izrecno imenovan Jupitrov orel, izpostavlja antično konotacijo, je prevladujoča simbolika krščanska. Grška in rimska ikonografija orla namreč ne po znata motiva pomladitve z letom proti soncu, ki orlu sežge stara peresa, da odpadejo in namesto njih zrastejo nova. Šele v renesančni emblematiki pride do povezovanja Jupitrovega orla, ki se v letu najbolj približa soncu, z motivom pomladitve, ki izhaja iz Fiziologa in svetopisemske tradicije. Pictura Valvasorjevega emblema posebej izposta vi epizodo z ožganimi peresi, ki odpadajo z orlovih kril. Motiv je v emblematiki dobro znan, embleme s to vsebino pa najdemo tako v Picinellijevi zbirki (I, 4, 160) kot v Bo schevi enciklopediji. Res je, da je izpadanje peres večkrat upodobljeno v trenutku, ko se orel približa soncu (npr. Claude Paradin, Devises Heroïques, Lyon 1551, 172; Bosch III, embl. 39), čeprav je tudi različica z orlom, ki leti stran od sonca, dokaj pogosta (npr. Camerarius, III, embl. 14; Bosch, I, embl. 853).44 202 NE GERM / SIMBOLIKA ORLA IN SONCA V EMBLEMIH SPOMINSKE KNJIGE LJUBLJANSKE PLEMIŠKE DRUŽBE SV. DIZMA ika 11: Cristoph Georg Eimmart, Emblem Formosus renascens, Wolfgang Helmha von Hohberg, Lust- und Artzeney-Garten des Königlichen Propheten Davids, Regensburg 1675, fol. 103r. lika 11: Cristoph Georg Eimmart, Emblem Formosus renascens, Wolfgang Helmhar von Hohberg, Lust- und Artzeney-Garten des Königlichen Propheten Davids, Regensburg 1675, fol. 103r. 45 »[…] pietas Divina suos licet ardor adurat/ igne crucis tamen has sic renovare solet.« Čeprav je v motivu orlovega preroda Kristus največkrat simbolično predstavljen s soncem, je tudi interpretacija orla kot podobe Križanega uveljavljena že v srednjeveški ikonografiji. Povezava ne temelji samo na ideji odrešitve in vnebovzetja, krščanski pisci namreč silhueto orla na nebu primerjajo tudi s križem. Glej na primer: Arnold Bonnevalski, Tractatus de septem verbis Domini in cruce, 7, PL 189, 1682. 2 Orel in sonce v emblemih članov Akademije Zedinjenih Slika 11: Cristoph Georg Eimmart, Emblem Formosus renascens, Wolfgang Helmhard von Hohberg, Lust- und Artzeney-Garten des Königlichen Propheten Davids, Regensburg 1675, fol. 103r. 203 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Dejstva, da je motiv orla z izpadajočimi peresi, ki leti proč od sonca, v emblematiki ustaljen ikonografski motiv, na tem mestu niti ni treba posebej dokazovati. Najdemo lahko celo prototip, po katerem se Valvasorjev emblem neposredno zgleduje. V že omenjeni Hohbergovi knjigi je na fol. 103r emblem, ki ima identično geslo kot Valva sorjev (Formosus renascens) in sličico, ki jo je Grahovar kompozicijsko skoraj v celoti povzel (slika 11). Primerjava pojasni tudi, zakaj se v emblemu Karla Jožefa Valvasorja pojavi dopolnilno besedilo: baron je očitno želel vključiti del Hohbergovega epigrama in Grahovar je vestno prepisal prva dva verza. Ikonografijo Valvasorjevega emblema dodatno osvetljujeta Hohbergova verza, ki sta v ljubljanski Spominski knjigi izpuščena: antično vsebino dopolnjujeta s krščanskim motivom preroda, ki je v izvirniku povezan z odrešenjem s Kristusovo smrtjo na križu.45 Kot v vseh predstavljenih emblemih se tudi tukaj heraldični orel preseli v emblemsko sličico in simbolično predstavlja samega naročnika. S posebej izpostavljeno antično ikonografijo orla kot Jupitrove svete ptice Valvasor hkrati opozarja na svoje zanimanje za kulturo klasične antike, ki so jo imeli kranjski akademiki zelo v čislih. 3 Zaključek Deset emblemov z motivom orla in sonca, ki krasijo vpisne strani v Dizmovi kroniki, ima v vsebinskem pogledu veliko skupnega, vendar so z ikonografskega vidika pose bej zanimive prav razlike, ki so včasih očitne, spet drugič v duhu žlahtne tradicije re nesančnega emblema spretno prikrite in zahtevajo dovolj pozornega bralca. Tovrstni specifični simbolni poudarki in duhovite domislice najbolje ilustrirajo inventivnost naročnikov ter pričajo o njihovem poznavanju evropske tradicije emblemskih knjig. Težnja po izvirnosti je še posebej razvidna, ko gre za oblikovanje individualne note emblemskega sporočila, s katero so želeli kranjski akademiki v Spominski knjigi pu stiti oseben pečat. Ikonološka analiza izbranih emblemov ne razkriva samo njihovega vsebinskega bogastva in kompleksne strukture, različne zglede in neposredne vplive, ampak hkrati celoviteje osvetljuje dejaven pristop naročnikov. Ob tem opozarja tudi na vlogo miniaturistov, ki nikakor niso bili samo izvrševalci naročil, temveč so ak tivno sodelovali pri sami ikonografski zasnovi emblemov. S tem se odpira vpogled v dinamiko odnosov med umetnikom in naročnikom, ki doslej v primeru ilustracij v Spominski knjigi ljubljanske plemiške družbe sv. Dizma ni bila deležna ustrezne po zornosti. Odgovori na vprašanja, ki si jih strokovna javnost do nedavnega sploh ni 204 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma zastavljala, sprožajo niz novih vprašanj. Kdaj in če sploh kdaj bomo našli odgovore nanje, je nemogoče napovedati. Bliskovit razvoj raziskav na področju emblematike, mednarodni raziskovalni projekti univerz in umetnostnih inštitutov, vse večja dosto pnost primarnih gradiv v digitalizirani obliki in odkrivanje novih emblemskih knjig vsekakor obetajo, da bomo v doglednem času tudi o emblemih kranjskih akademikov vedeli še precej več kot danes. Viri Ajlijan, Perí zóion idiótetos (De natura animalium). Dostopno z angleškim prevodom A. F. Scholfielda: Aelian, On the Characteristics of Animals, 3 vols. (The Loeb Classical Library), i Harvard University Press, Cambridge, Mass. 1958–59. ih Harvard University Press, Cambridge, Mass. 1958–59. Alciati, Andrea, Emblematum liber, Heinrich Steyner, Augsburg 1531. Aldrovandi, Ulisse, Ornithologiae, hoc est de avibus historiae libri XII, Apud Franciscum de Franciscis Senensem, Bologna 1599. Ambrož Milanski, Hexaemeron, Migne, PL 14, 232. Ambrož (Psevdo), Sermo 46 de Salomone, Migne, PL 17, 718. Apes aCaDeMICae Operosorum Labacensium, sive Institutum, leges, scopus, nomina, et symbola novae Academiae sub apum symbolo Labaci adunatae: orbi literario exhibitae cum oratione inaugurali in primo conventu publico ad proceres Aemonae dicta, Janez Jurij Mayr, Ljubljana 1701. Aristotel, Tón perí tá zóia istoríon (Historia animalium). Dostopno z angleškim prevodom A. L. Pecka: Aristotle, History of Animals (The Loeb Classical Library), Harvard University Press, Cambridge, Mass. 1965. Arnold Bonnevalski, Tractatus de septem verbis Domini in cruce, 7, Migne, PL 189, 1682. Avguštin Hiponski, Enarrationes in Psalmos, Migne, PL 37. Avguštin Hiponski, In Evangelium Joannis tractatus, Migne, PL 35, 1379–1976. Avguštin (Psevdo), Meditationum Liber Unus, Migne, PL 40, 901–942. Bartolomej Anglež, De proprietatibus rerum, Johannes Veldener, Köln 1471. Bosch, Jacob, Symbolographia sive De arte symbolica sermones septem …, Johann Caspar Ben card, Augsburg/Dilingen 1702. Camerarius, Joachim, Symbolorum et emblematum ex animalibus quadrupedibus desumtorum centuria altera collecta, P. Kaufmann, Nürnberg 1595. Camerarius, Joachim, Symbolorum & emblematum ex volatilibus et insectis desumtorum centuria tertia collecta, P. Kaufmann, Nürnberg 1596. Camerarius, Joachim, Symbolorum et emblematum ex aquatilibus et reptilibus desumptorum cen turia quarta, Typis Voegelinianis, Leipzig 1604. Dolničar, Janez Gregor, Konceptna knjiga, Semeniška knjižnica, Ljubljana, sign. Rokopis 6. Ezop, Kritična izdaja ezopovskih basni je dostopna z angleškim prevodom B. E. Perryja: Babrius and Phaedrus (The Loeb Classical Library), Harvard University Press, Cambridge, Mass. 1965. h Fiziolog, Physiologos. Le bestiaire des bestiaires, Texte traduit du grec, établi et commenté par Arnaud Zucker, Éditions Jérôme Millon, Paris 2005. 205 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Gessner, Conrad, Historiae animalium liber III., qui est de avium natura, Christoph Froschove rus, Zürich 1555. Giovio, Paolo, Dialogo dell'imprese militari et amorose, Antoine Barré, Rim 1555. Gregor Veliki, Moralium Libri Sive Expositio In Librum Beati Job, II, Migne, PL 76. Guillaume Le Clerc, Bestiaire divin, sredina 13. st., Bodleian Library, MS. Douce 132. Viri 206 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Ruscelli, Girolamo, Imprese illustri …, Francesco Rampazetto, Benetke (vol. I–III. 1572, Libro quarto 1583). Schönleben, Janez Ludvik, Feyertäglicher Erquick-Stunden: Das ist: Ehren- vnd Lobpredigen der lieben Heiligen Gottes … Anderer Theil, Melchior Haan, Salzburg 1670. h Sveto pismo Stare in Nove zaveze, Slovenski standardni prevod, Svetopisemska družba Slovenije, Ljubljana 1996. Theatrum Memoriae Nobilis ac Almae Societatis Unitorum das ist Schau Bühne der Gedächtnuß der Adelichen und Gottseeligen Geselschafft der Vereinigten zu stätts wherenden Andenken eröffnet in der Uhralten Haubt Statt Laybach, Ljubljana 1688–1801, Arhiv Republike Slovenije, AS 1073, I/1. Vergilij, Georgike, Ekloge, Eneida. Dostopno z angleškim prevodom H. R. Fairclougha: Virgil, Eclogues. Georgics. Aeneid. Books I-VI, (The Loeb Classical Library), William Heinemann, London/ Harvard University Press, Cambridge, Mass. 1916. Vergilij, Eneida. Dostopno z angleškim prevodom H. R. Fairclougha: Virgil, Aeneid, Books VII -XII, Appendix Vergiliana (The Loeb Classical Library), William Heinemann, London/ Harvard University Press, Cambridge, Mass. 1918. Zani, Valerio, Memorie, Imprese, e Ritratti de’ Signori Accademici Gelati de Bologna …, Manolesi, Bologna 1672. Viri Hieronim, Breviarium in Psalmos, Migne, PL 26, 1097. Hohberg, Wolfgang Helmhard von, Lust- und Artzeney-Garten des Königlichen Propheten Da vids…, Christoph Fischer, Regensburg 1675. p g g Homer, Iliada, prev. A. Sovre, Državna založba Slovenije, Ljubljana 1982. Horacij, Ode. Dostopno z angleškim prevodom N. Rudda: Horace, Odes and Epodes, (The Loeb Classical Library), Harvard University Press, Cambridge, Mass. 2004. Izidor Seviljski, Ethymologiarum sive Originum liber XII. Dostopno v zbirki Latin Library: http:// www.thelatinlibrary.com/isidore/12.shtml. Klavdijan, Panegyricus Manlio Theodoro consuli. Dostopno z angleškim prevodom M. Platna uerja: Claudian vol. I, Panegyric on Probinus and Olybrius, Against Rufinus 1 and 2. War Against Gildo, Against Eutropius 1 and 2, Fescennine Verses on the Marriage of Honorius, Epithalamium of Honorius and Maria, Panegyrics on the Third and Fourth Consulships of Honorius, Panegyrics on the Consulship of Fl. Manlius Theodorus, On Stilicho's Consulship (The Loeb Classical Library), Harvard University Press, Cambridge, Mass. 1922. Le Brun, Laurentius, Virgilius Christianus, I. Liber Fastorum, sive Hexaemeron & de opere sex dierum, Simeon Piget, Paris 1661. Lukan, Mark Anej, Pharsalia (De bello civilo). Dostopno z angleškim prevodom J. D. Duffa: The Civil War Books I-X (Pharsalia), (The Loeb Classical Library), William Heinemann, Lon don/ Harvard University Press, Cambridge, Mass. 1928. Ovidij (Publij Ovidij Nazon), Metamorphoses. Dostopno z angleškim prevodom F. J. Millerja: Ovid, Metamorphoses, 2 vols. (Loeb Classical Library), Harvard University Press, Cambrid ge, Mass. 1916. g Ovidij (Publij Ovidij Nazon), Metamorfoze I.–III., prev. Barbara Šega Čeh, Modrijan, Ljubljana 2013. Picinelli, Filippo, Mundus symbolicus: in emblematum universitate formatus, explicatus, et tam sacris quàm profanis …, Hermann Demen, Köln 1681. ̀ Pindar, Pitijske ode, Olimpijske ode. Dostopno z angleškim prevodom H. Racea: Pindar, Olym pian Odes. Pythian Odes, vol. I (Loeb Classical Library), Harvard University Press, Cam bridge, Mass. 1997. Pindar, Istmijske ode. Dostopno z angleškim prevodom H. Racea: Pindar, Nemean Odes. Isthmi an Odes. Fragments, vol. II (Loeb Classical Library), Harvard University Press, Cambridge, Mass. 1997. Pittoni, Giovanni Battista, Imprese di diversi prencipi, duchi, signori, e d'altri personaggi et huo mini letterati et illustri …, Benetke 1562. Plinij Starejši, Naturalis historia libri XXXVII. Dostopno z angleškim prevodom H. Rackhama: Pliny the Elder, Natural History, 10 vols. (The Loeb Classical Library), Harvard University Press, Cambridge, Mass. 1947. g Ripa, Cesare, Iconologia, Appresso Lepido Facij, Rim 1603. Ripa, Cesare, Iconologia, Appresso Lepido Facij, Rim 1603. Rollenhagen, Gabriel, Nucleus Emblematum selectissimorum …, Crispijn de Passe, Köln 1611. Strokovna literatura Baraga, France, Dolničarjeva Konceptna knjiga ljubljanske plemiške družbe sv. Dizma, Uvodna pojasnila, v: Spominska knjiga ljubljanske plemiške družbe sv. Dizma 1688–1801, I (ur. Go stiša, L.), Fundacija J. V. Valvasorja, Ljubljana 2001, str. 235–236. Cevc, Emilijan, Iluminirani kodeks ljubljanske plemiške družbe sv. Dizma v: Spominska knjiga ljubljanske plemiške družbe sv. Dizma, 1688–1801, II (ur. Gostiša, L.), Fundacija J. V. Valva sorja, Ljubljana, 2001, str. 67–113. Dolinar, France Martin, Od Dizmove bratovščine do Akademije delovnih v Ljubljani, v: Acade mia Operosorum, zbornik prispevkov s kolokvija ob 300-letnici ustanovitve, (ur. Gantar, K.), SAZU, Ljubljana 1994, str. 35–46. Germ, Martin, Iconography of Emblematic Animals in the Album of the Ljubljana Noble Socie ty of St Dismas: Readings and Misreadings, IKON Journal of Iconographic Studies, 2 (2009), str. 305–312. Germ, Tine, Spominska knjiga ljubljanske plemiške družbe sv. Dizma: zgodovinski kontekst nastanka in njeni idejni vzori. Arhivi, 34/1 (2011), str. 33–40.i Germ, Tine, Smrt kraljuje povsod in bela Smrt triumfira: Valvasorjevo Prizorišče človeške smrti v evropskem kontekstu, Znanstvena založba FF, Ljubljana 2015.h Germ, Martin, The emblems of the Album of the Ljubljana Noble Society of St Dismas: Context, Sources, Originality, Ars & humanitas, 11/1 ( 2017), str. 149–170. Golob, Nataša, Spominska knjiga ljubljanske plemiške družbe sv. Dizma. Kodikološke ugotovi tve, v: Spominska knjiga ljubljanske plemiške družbe sv. Dizma 1688–1801, II (ur. Gostiša, L.), Fundacija J. V. Valvasorja, Ljubljana 2001, str. 41–47. Gradel, Ittai, Emperor Worship and Roman Religion, OUP, Oxford 2002. Henkel, Arthur; Schöne, Albrecht, Emblemata. Handbuch zur Sinnbildkunst des XVI. und XVII. Jahrhunderts, J. B. Metzler, Stuttgart 1967. 207 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Kastelic, Jože, Emblemi v ikonografskem ogledalu. Katalog, v: Spominska knjiga ljubljanske ple miške družbe sv. Dizma 1688–1801, II (ur. Gostiša, L.), Fundacija J. V. Valvasorja, Ljubljana 2001, str. 119–261. Lavrič, Ana, Societas Unitorum – Akademija Združenih, v: J. G. Dolničar, Zgodovina ljubljanske stolne cerkve, (ur. Lavrič, A.), Založba ZRC, Ljubljana 2003, str. 27–30. Lisac, Ljubomir Andrej; Reisp, Branko, s. v.: Trost, Andrej, Slovenski biografski leksikon, IV, Lju bljana, 1960, str. 186–187; http://www.slovenska-biografija.si/oseba/sbi724835/. i Miklavčič, Maks, s. v.: Schönleben, Janez Ludvik, Slovenski biografski leksikon, X, Ljubljana 1967; http://www.slovenska-biografija.si/oseba/sbi548709/. i Panofsky Ervin, Hercules am Scheidewege und andere antike Bildstoffe in der neuen Kunst (Stu dien der Bibliothek Warburg, Band 18), Teubner Verlag, Berlin 1930. Schnabel, Werner Wilhelm, Das Stammbuch. Strokovna literatura Konstitution und Geschichte einer textsortenbez ogenen Sammelform bis ins erste Drittel des 18. Jahrhunderts, Niemeyer, Tübingen 2003. Smolik, Marijan, Pregled članstva ljubljanske plemiške družbe sv. Dizma, v: Spominska knjiga ljubljanske plemiške družbe sv. Dizma 1688–1801, II (ur. Gostiša, L.), Fundacija J. V. Valva sorja, Ljubljana 2001, str. 263–275. Vignau-Wilberg, Thea, Archetypa studiaque patris Georgii Hoefnagelii, 1592: Natur, Dichtung und Wissenschaft in der Kunst um 1600, Staatliche Graphische Sammlung, München 2005. t Vidmar, Luka, Ljubljana kot novi Rim. Akademija Operozov in baročna Italija, Založba ZRC, Ljubljana 2013. Vrhunc, Polonca, Simon Tadej Volbenk Grahovar (1710–1774), Zbornik za umetnostno zgodo vino, n. v. 8 (1970), str. 107–132. Tine Germ Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma Ključne besede: Spominska knjiga ljubljanske plemiške družbe sv. Dizma, renesančna in baročna emblematika, simbolika orla in sonca, knjižna iluminacija, Janez Gregor Dolničar, Andrej Trost, Simon Tadej Volbenk Grahovar Avtor v svoji študiji analizira ročno slikane embleme z motivom orla in sonca v Spominski knjigi ljubljanske plemiške družbe sv. Dizma (skrajšano: Dizmova kronika), hranjene v Arhivu Republi ke Slovenije (ref. AS 1073, I/1), ki so z ikonografskega vidika skoraj povsem neraziskani. Kratke mu uvodu, v katerem je predstavljena Dizmova kronika kot izvirna oblika emblemske spomin ske knjige, ki je nastajala v Ljubljani med letoma 1689 in 1801, sledi ikonološka analiza desetih emblemov z motivom orla in sonca (fols. 119r, 161r, 229r, 234r, 238r, 264r, 287r, 294r, 306r, 369r) v kontekstu evropske emblematike. Avtor najprej opozori na ikonografska izhodišča mo tiva ter opredeli osnovni vsebinski okvir, ki je skupen vsem emblemom z motivom orla in son ca v Dizmovi kroniki. Gre za večinoma kristološko ikonografijo, ki izhaja iz biblijske eksegeze, Fiziologa in srednjeveških bestiarijev, največji razcvet pa doživi prav na področju emblematike 208 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma v zgodnjem novem veku. Skupno vsebinsko jedro oblikujejo motivi preroda, odrešenja in več nega življenja ter simbolične predstave o sledenju Kristusovemu nauku in odličnosti duha, ki se dviga v nebeške višave. Analiza pokaže, da imajo ključno vlogo motivi, ki izvirajo iz antike, a so v krščanstvu dobili nove, precej bolj kompleksne vsebine: orel, ki nemoteno zre v sonce, orel, ki leti proti soncu, preizkušnja mladih orličev z gledanjem v sonce ter orel, ki uči mladiče leteti. Raziskava se osredotoča na ikonografsko specifiko vsakega posameznega emblema, v ka teri sta razvidna inventivnost naročnikov in njihovo poznavanje evropske tradicije emblemskih knjig. Analiza izbranih emblemov ne razkriva samo njihovega vsebinskega bogastva in komple ksne strukture, različne zglede in neposredne vplive, ampak hkrati celoviteje osvetljuje dejaven pristop naročnikov v procesu njihovega nastajanja, ki doslej v primeru ilustracij v ljubljanski Spominski knjigi ljubljanske plemiške družbe sv. Dizma ni bil deležen ustrezne pozornosti. Ob tem avtor opozarja tudi na vlogo vodilnih miniaturistov Andreja Trosta in Simona Tadeja Vol benka Grahovarja, ki nista bila zgolj iluminatorja, temveč sta aktivno sodelovala pri sami iko nografski zasnovi emblemov, s čimer odpira nov vpogled v dinamiko odnosov med umetnikom in naročnikom. Tine Germ Tine Germ The Eagle and the Sun Symbolism in the Emblems of the Album of the Ljubljana Noble Society of St Dismas Keywords: The Album of the Ljubljana Noble Society of St Dismas, Renaissance and Baroque emblems, the eagle and the sun symbolism, manuscript illumination, Janez Gregor Dolničar, Andrej Trost, Simon Tadej Volbenk Grahovar The paper provides an analysis of the hand-painted emblems with the motifs of the eagle and sun in the Album of the Ljubljana Noble Society of St Dismas (the Archive of the Republic of Slo venia, ref. AS 1073, I/1). These have so far barely received any iconographic interpretation, nor have there been any serious attempts to identify the potential sources or models. A short intro duction points to the original form of the Ljubljana Album as a memorial book with individual emblems created for the members of the Society of St Dismas between 1689 and 1801. The focus of the article is on the iconological analysis of ten emblems with the motifs of the eagle and the sun (the Ljubljana Album, fols. 119r, 161r, 229r, 234r, 238r, 264r, 287r, 294r, 306r, 369r) in the context of European emblematics. The author draws attention to the iconographic specifics of each individual emblem, as well as to the basic symbolism of the eagle and sun which is com mon to all of them. While the common iconography of the motif in the selected emblems in the Ljubljana Album regularly relates to the Christological contexts of the eagle and the sun symbolism as developed in the biblical exegesis, Physiologus, the medieval bestiaries and various theological writings, the individual allegorical message depends on the complex interaction of visual and textual components of each emblem. The traditional Christological topics of renewal through strong faith and repeated purification of body and mind, the ascending of the Spirit into higher 209 ne Germ / Simbolika orla in sonca v emblemih Spominske knjige ljubljanske plemiške družbe sv. Dizma realms, the salvation of the soul in Christ and the eternal life in Heaven, are often completed with themes which originate in Classical mythology and ancient animal lore. Some motifs that combine both Christian and Classical symbolism of the eagle and the sun appear to have been particularly appreciated among the members of St Dismas Society: the eagle looking unblinking into the sun, the eagle flying towards the sun, the trial of eaglets gazing at the sun, and the eagle teaching eaglets to fly. O avtorju Tine Germ je redni profesor na Oddelku za umetnostno zgodovino Filozofske fakultete Univer ze v Ljubljani, strokovnjak za ikonografijo in ikonologijo poznega srednjega in zgodnjega no vega veka. Njegove raziskave se osredotočajo na humanistične teme v renesančni umetnosti, na renesančno in baročno emblematiko ter ikonografijo živali v umetnosti zgodnjega novega veka. The Eagle and the Sun Symbolism in the Emblems of the Album of the Ljubljana Noble Society of St Dismas The iconographic particularities of each emblem show the erudition of the patrons as well as their knowledge of European books of emblems. The analysis of the assort ed emblems goes beyond mere presentation of their rich content and complex structure, their various models and influences; it also brings an in-depth look at the patrons’ active involvement in the creative process which, in the case of the Album of the Ljubljana Noble Society of St Dis mas, has not yet been dealt with properly. It furthermore draws attention to the role played by the two leading miniaturists Andrej Trost and Simon Tadej Volbenk Grahovar, who occasionally advised the patrons on both visual and iconographic components of the emblems, thus acting not only as illuminators but also as creators of the emblems. About the author Tine Germ is a Professor at the Department of Art History, Faculty of Arts, University of Lju bljana, an expert in iconography and iconology of Late Mediaeval and Early Modern Art. His research work focuses on humanistic themes in Renaissance Art, Renaissance and Baroque em blems, and animal symbolism in Early Modern Art. 210
https://openalex.org/W2973788067	https://europepmc.org/articles/pmc6754425?pdf=render	English	null	Chromosomal instability of circulating tumor DNA reflect therapeutic responses in advanced gastric cancer	Cell death and disease	2,019	cc-by	6,005	© The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Abstract Gastric cancer is characterized by chromosomal instability. In this study, we investigated chromosomal instability quantiﬁed by copy number instability (CNI) score of circulating tumor DNA (ctDNA) during the drug treatment in advanced gastric cancer (AGC). A total of 55 pretherapeutic plasmas from 55 AGC patients and 75 plasmas during drug treatment of 26 AGC patients were collected. Plasma ctDNA was extracted and assessed by whole-genome sequencing (WGS) for somatic copy number alteration (SCNA), and according to which we calculated the CNI scores. We next assessed the correlations between chromosomal instability and therapeutic response. The cutoff value of chromosomal instability was deﬁned as the mean + SD of the CNI scores (56.60) in cfDNA of plasmas from 100 healthy people. For 55 enrolled cases, chromosomal instability was observed in 27 (49%) prior to drug treatment, whose response rate (59%, 16/27) was higher than in 28 patients with stable chromosomes (32%, 9/28, P = 0.043). We also observed that CNI scores ﬂuctuated during treatment in 26 patients. Speciﬁcally, the CNI scores in 93% (14/15) of patients sensitive to drug treatment reduced to the level of chromosomal stability and the CNI scores in 52% (13/25) of patients resistant to treatment elevated again. For ctDNA with developed resistance, the SCNA patterns were identical to those before treatment, whereas the CNI scores were lower than the pretherapeutic scores. We found that chromosomal instability based on ctDNA could predict and monitor therapeutic response in gastric cancer, although validation in a larger cohort will be necessary. A R T I C L E O p e n A c c e s s Chromosomal instability of circulating tumor DNA reﬂect therapeutic responses in advanced gastric cancer Cheng Zhang1, Mengqi Zhang1, Beifang Li1, Yunyun Niu2, Limeng Chen2, Jing Yang1, Sijia Lu2, Lin Shen1 Zuhua Chen1, Cheng Zhang1, Mengqi Zhang1, Beifang Li1, Yunyun Niu2, Limeng Chen2, Jing Yang1, Sijia Lu2, Jing Gao1 and Lin Shen1 Chen et al. Cell Death and Disease (2019) 10:697 https://doi.org/10.1038/s41419-019-1907-4 Chen et al. Cell Death and Disease (2019) 10:697 https://doi.org/10.1038/s41419-019-1907-4 Cell Death & Disease Cell Death & Disease Introduction targets for GC remains limited3. Although several land- mark studies have highlighted the molecular subtypes of GC on DNA, RNA, and protein levels based on large- cohort tumor tissues, the application of such markers in clinical practice remains to be a big challenge4–6. Although predictive and resistance markers are equally important for systemic antitumor therapy, most research efforts to date have been focused on exploring the efﬁcacy of predictive markers, while only a few clear markers of resistance have been found7,8. Meanwhile, due to the speciﬁc features of GC, the current Response Evaluation Criteria in Solid Tumors (RECIST) fail to evaluate drug resistance in a precisely and timely manner9. Gastric cancer (GC) is featured by the high hetero- geneity on anatomical, molecular, and cellular levels, which greatly impeded the current therapeutic develop- ment for advanced GC (AGC)1,2. Individualized che- motherapeutic or targeted therapy guided by predictive and monitoring markers has been progressed desirably into the era of precision medicine, yet the therapeutic Correspondence: Sijia Lu (lusijia@yikongenomics.com) or Jing Gao (gaojing_pumc@163.com) or Lin Shen (linshenpku@163.com) 1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute, Beijing, China 2Department of Clinical Research, Yikon Genomics Co. Ltd., Shanghai, China These authors contributed equally: Zuhua Chen, Cheng Zhang Edited by Y. Shi Chromosomal instability, often indicated as large or small somatic gains and losses on chromosomal level, has © The Author(s) 2019 © The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) Ofﬁcial journal of the Cell Death Differentiation Association Chen et al. Cell Death and Disease (2019) 10:697 Chen et al. Cell Death and Disease (2019) 10:697 Page 2 of 9 Table 1 The clinicopathological characteristics of patients (N = 55) Characteristics Number (%) Sex Male 43 (78.2) Female 12 (21.8) Age <65 41 (74.5) ≥65 14 (25.5) Tumor location EGJ 21 (38.2) Non-EGJ 34 (61.8) Differentiation High 4 (7.3) Middle 23 (41.8) Low 28 (50.9) Lauren classiﬁcation Intestinal type 38 (69.1) Diffuse type 10 (18.2) Mixed type 7 (12.7) HER2 status Positive 30 (54.5) Negative 25 (45.5) Targeted treatment Yes 30 (54.5) No 25 (45.5) Table 1 The clinicopathological characteristics of patients (N = 55) been widely accepted as a hallmark of cancer10. A large amount drugs exert their antitumor activities through disrupting chromosomal stability11. Moreover, chromo- somal instability has been reported to drive the clonal evolution of intratumoral heterogeneity, and confer the intrinsic and acquired resistance11,12. Hence, dynamically deciphering the landscape of chromosomal stability dur- ing treatment is of great importance in help under- standing the therapeutic effect as well as cancer evolution. Nevertheless, the sequential acquisition of tumor tissues is clinically impractical, and new approach to achieve sur- veillance for patients’ response is urgently demanded. With the rapid progress of high-throughput sequencing and liquid biopsy techniques, circulating tumor DNA (ctDNA) presented in plasma have become the alternative surrogate to tissues13. Genome-wide proﬁling of copy number instability (CNI) in ctDNA provides a suitable method to quantify chromosomal instability and predict therapeutic response in several cancers14,15. However, the signiﬁcance and dynamic changes of chromosomal instability during the treatment of AGC have not been reported. The clinicopathological characteristics of 26 patients Yes The clinicopathological characteristics of 26 patients A total of 55 patients were enrolled with a median age of 58 years (range: 29–80 years). Totally, 43 patients (78%) were male and 12 (22%) were female. Most of the patients were diagnosed with HER2-positive (55%, 30/55), poorly differentiated (51%, 28/55), and intestinal (69%, 38/55) AGC. All of the patients received at least two cycles of drug treatment with 30 patients receiving targeted therapy (pyrotinib, RC48, trastuzumab, pertuzumab, and fuquin- tinib) alone or in combination with chemotherapy and 25 patients undergoing chemotherapy alone. The detailed clinicopathological characteristics of 55 patients are shown in Table 1 and Table S1. genes included ERBB2 (17q21), MYC (8q24), GNAS (20q13), EGFR (7p11), ZNF217 (20q13), CCNE1(19q12), NCOA3 (20q13), and CDK6 (7q21), and the most fre- quently deleted genes were CDKN2A (9p21), CDKN2B (9p21), KIT (4q12), SMAD4 (18q21), and FGFR1 (8p12). Most of these genes encoded receptor tyrosine kinases and cell cycle-related proteins, which was consistent with the previously published studies on GC4,17. © The Author(s) 2019 Our previous research indicated that the HER2 copy number detected by plasma ctDNA could serve as a response biomarker of trastuzumab treatment16. In the present study, we attempted to elucidate the chromoso- mal instability based on ctDNA during chemotherapy or targeted therapy by whole-genome sequencing and investigate their signiﬁcance in AGC. CNI scores of ctDNA in all patients Genome-wide patterns of SCNA detected in ctDNA derived from baseline plasma and paired blood samples showed high tumor heterogeneity among patients (Fig. 1 and Fig. S1). According to the proﬁling of segmented copy numbers, large-scale copy number gain or loss was observed on chromosomes 5, 7, 8, 13, 17, 18, 19, and 20 in ctDNAs of plasmas 5001, 7335, 5185, 8001, 5978, and 7294. Among the tumor-related genes, the top ampliﬁed We calculated the CNI scores in ctDNA from 130 plasmas from 55 patients and cfDNA from 100 plasmas from 100 healthy people (Table S2). The cutoff value of the chromosomal instability was deﬁned as the mean + SD of the CNI scores (56.60) in 100 plasmas from healthy people. Chromosomal instability with diverse patterns was observed in 27 of 55 patients (49%) prior to drug treatment. Ofﬁcial journal of the Cell Death Differentiation Association Chen et al. Cell Death and Disease (2019) 10:697 Page 3 of 9 Fig. 1 SCNV patterns of ctDNA at baseline among 26 patients. Genome-wide patterns of somatic copy number variation (SCNV) based on ctDNA of baseline plasma and corresponding blood cell samples from 26 patients were analyzed and plotted. The x-axis and y-axis represent the loci of 22 chromosomes and corresponding copy numbers, respectively. CNI score copy number instability score me-wide patterns of somatic copy number variation (SCNV) based on ctDNA nts were analyzed and plotted. The x-axis and y-axis represent the loci of 22 Fig. 1 SCNV patterns of ctDNA at baseline among 26 patients. Genome-wide patterns of somatic copy number variation (SCNV) based on ctDNA of baseline plasma and corresponding blood cell samples from 26 patients were analyzed and plotted. The x-axis and y-axis represent the loci of 22 chromosomes and corresponding copy numbers, respectively. CNI score copy number instability score Among 26 patients with paired blood cells and dynamic plasma samples, the CNI scores of ctDNA derived from baseline plasmas were signiﬁcantly higher than those from paired blood cells (67.62 ± 15.99 vs. 49.88 ± 2.47, P < 0.001) (Fig. 2a). The CNI scores after drug treatment were signiﬁcantly lower than they were before treatment (56.94 ± 15.50 vs. 67.62 ± 15.99, P < 0.001) (Fig. 2b). Moreover, CNI scores ﬂuctuated during therapy. CNI scores of ctDNA in all patients 93% (14/ 15) of post-therapeutic ctDNA at the time of partial response (PR) had the lowest scores (48.85 ± 2.54), while 52% (13/25) of ctDNA with developed resistance to therapy retained elevated scores (63.75 ± 18.65). For ctDNA with developed resistance, SCNA patterns were identical to those before treatment, but the instability scores were lower than the pretherapeutic level (63.75 ± 18.65 vs. 67.62 ± 15.99) (Fig. 2c). determined as SD by CT after four or six cycles’ treatment respectively, the tumor biomarkers (CEA, CA199, and CA72.4) were increased, which suggested a trend toward disease progression. For case 12 and case 22, the increase of tumor biomarkers at the time of SD was not signiﬁcant, and only CA72.4 increased slightly. Ofﬁcial journal of the Cell Death Differentiation Association Chromosomal instability of ctDNA prior to treatment could predict the therapeutic response Among patients with chromosomal instability prior to treatment (n = 27), 16 patients (59%) achieved PR after drug treatment, 10 patients (37%) achieved SD, and 1 patients (4%) achieved progressive disease (PD). For patients with chromosomal stability (n = 28) prior to treatment, 9 patients (32%) achieved PR after treatment, 16 patients (57%) achieved SD, and 3 patients (11%) achieved PD. The response rate (59%, 16/27) of patients with chromosomal instability was higher than that (32%, 9/28) of patients with chromosomal stability (P = 0.043) (Fig. 3 and Table S3). Five ctDNA from four patients at the timepoint of stable disease (SD) showed relatively high CNI scores (cases 3, 6, 12, and 22), which was analyzed individually (Fig. 2d). For case 3 and case 6, although the clinical response was Ofﬁcial journal of the Cell Death Differentiation Association Chen et al. Cell Death and Disease (2019) 10:697 Page 4 of 9 Fig. 2 The CNI scores of ctDNA in 26 patients with dynamic plasma samples. a The CNI scores of ctDNA in plasmas and paired blood cells from 26 AGC patients. b The CNI scores of ctDNA from plasma samples before and after treatment in 26 AGC patients. c The CNI scores of ctDNA from plasma under different clinical responses. CNI score copy number instability score, PR partial response, SD stable disease, PD progressive disease. All data are presented as mean ± SD. P < 0.001 according to t test or one-way ANOVA. d The dynamic changes of CNI scores and tumor biomarkers during the administration of treatments in four patients Fig. 2 The CNI scores of ctDNA in 26 patients with dynamic plasma samples. a The CNI scores of ctDNA in plasmas and paired blood cells from 26 AGC patients. b The CNI scores of ctDNA from plasma samples before and after treatment in 26 AGC patients. c The CNI scores of ctDNA from plasma under different clinical responses. CNI score copy number instability score, PR partial response, SD stable disease, PD progressive disease. All data are presented as mean ± SD. P < 0.001 according to t test or one-way ANOVA. d The dynamic changes of CNI scores and tumor biomarkers during the administration of treatments in four patients Dynamic changes in CNI scores of ctDNA could indicate disease progression patients are also further demonstrated in Fig. 5. For case 26, with chromosomal stability (score, 51.23) at baseline, achieved SD after treatment with gradually swelling ret- roperitoneal lymph nodes until the progressive of disease (Fig. 5a). For case 22 (score, 84.76) and case 18 (score, 87.10), which had high levels of chromosomal instability at baseline, distant metastases were signiﬁcantly shrunk Based on the clustering heatmap (Fig. 4), the CNI scores changed dynamically during therapy, which decreasing at PR or SD and re-increasing at PD. The detailed changes of CNI scores and therapeutic responses in 26 patients are shown in Figs. S2, S3, and Table S4. Four exemplary Ofﬁcial journal of the Cell Death Differentiation Association Chen et al. Cell Death and Disease (2019) 10:697 Page 5 of 9 Fig. 3 Schematic of 130 plasma samples and therapeutic response from 55 patients. For patients with pretherapeutic chromosomal instability, the response rate (59%, 16/27) was signiﬁcantly higher than that in patients with chromosomal stability (32%, 9/28) (P = 0.043). The cutoff value of chromosomal instability was deﬁned as the mean + SD of the CNI scores (56.60) in cfDNA from 100 healthy people. CNI scores also ﬂuctuated during drug treatment among 26 patients with dynamic plasmas. CNI scores copy number instability score, CIN chromosomal instability, CNS chromosomal stability, PR partial response, SD stable disease, PD progressive disease Fig. 3 Schematic of 130 plasma samples and therapeutic response from 55 patients. For patients with pretherapeutic chromosomal instability, the response rate (59%, 16/27) was signiﬁcantly higher than that in patients with chromosomal stability (32%, 9/28) (P = 0.043). The cutoff value of chromosomal instability was deﬁned as the mean + SD of the CNI scores (56.60) in cfDNA from 100 healthy people. CNI scores also ﬂuctuated during drug treatment among 26 patients with dynamic plasmas. CNI scores copy number instability score, CIN chromosomal instability, CNS chromosomal stability, PR partial response, SD stable disease, PD progressive disease copy number alteration (SCNA) pattern is a more uni- versal approach based on chromosomal instability13,15. With the next-generation sequencing of ctDNA applied to the quantitative analysis of the SCNA pattern, it provides a sufﬁcient method for early diagnosis of cancer15,20, prediction of treatment response14, and dynamic mon- itoring of acquired resistance19. with decreased CNI scores, and when patients developed resistance to treatment, the scores increased again (Fig. 5b, c). Dynamic changes in CNI scores of ctDNA could indicate disease progression For case 21, with chromosomal stability (score, 54) at baseline, liver metastases increased rapidly after treat- ment, and the CNI score increased signiﬁcantly (Fig. 5d). Changes in the CNI scores of these patients were observed during therapy (Fig. 5e). GC is featured by frequent SCNA and high- chromosomal instability. Among 295 patients with pri- mary gastric adenocarcinomas from The Cancer Genome Atlas (TCGA) cohort, 50% showed chromosomal instability4. In the present study, chromosomal instability detected by whole ctDNA-based genome sequencing was observed in 27 of 55 patients (49%) prior to drug treat- ment. No signiﬁcant relationship was found between patients’ characteristics and CNI scores, and we also did not ﬁnd differences between survival and CNI scores (data not shown). It seemed that the SCNA patterns of ctDNA with developed resistance were similar to those of paired pre- therapeutic ctDNA (Fig. 5). A circular map veriﬁed the identity of the SCNA pattern between pretherapeutic and resistant ctDNA, but the CNI score at resistance was lower than the pre-therapeutic level. Three representative patients are shown in Fig. 6. Discussion Chromosomal instability has been reported to underpin intratumoral heterogeneity, accelerate clone evolution, drive phenotypic adaptation, ﬁnally resulting in a poor clinical outcome, and accelerating therapeutic resistance in various cancers11,18,19. Compared with single- nucleotide polymorphism, the analysis of the somatic Chromosomal instability has been demonstrated to predict therapeutic response to radiotherapy, che- motherapy, and immunotherapy14,21,22. Recently, researchers observed a prediction accuracy of 83% of the Ofﬁcial journal of the Cell Death Differentiation Association Chen et al. Cell Death and Disease (2019) 10:697 Page 6 of 9 Fig. 4 Cluster analysis of SCNV patterns and therapeutic response among 26 patients. In the dynamic administration of treatment, the CNI scores showed a sharp decline at PR, then achieved slow recovery at SD, and ﬁnally increased signiﬁcantly at PD. The x-axis and y-axis represent the chromosomes’ loci and corresponding normalized copy numbers in plasma samples. PR partial response, SD stable disease, PD progressive disease, CN normalized copy number Fig. 4 Cluster analysis of SCNV patterns and therapeutic response among 26 patients. In the dynamic administration of treatment, the CNI scores showed a sharp decline at PR, then achieved slow recovery at SD, and ﬁnally increased signiﬁcantly at PD. The x-axis and y-axis represent the chromosomes’ loci and corresponding normalized copy numbers in plasma samples. PR partial response, SD stable disease, PD progressive disease, CN normalized copy number declined at PR and distinctly increased at PD. Compared with the traditional biomarkers, the CNI score elevated in 10/14 patients at progressive disease, accompanied by increasing levels of CEA, CA19-9, CA125, and CA72.4, alone or in combination (Fig. S4). More importantly, for case 22 with pre-therapeutic chromosomal instability, although the tumor biomarkers were decreased at stable disease (evaluated by RECIST criteria) after three cycles of treatment, we observed sharp increase of CNI score (44.28–91.41). This suggested that ctDNA-based CNI scores could serve as an early indicator of progression disease, whose power was comparable to the combination of CEA, CA19-9, CA125, and CA72-4. quantiﬁed chromosomal instability and the prediction of SD vs. PD prior to standard imaging analysis14. In our research, we found that pre-treatment chromosomal instability could predict a higher response rate (59% vs. 32%, P = 0.043). Compared with baseline, the scores of 19 patients (73%) decreased after drug treatment, and greatly increased when disease progressed, to a level slightly lower than the baseline value. Ofﬁcial journal of the Cell Death Differentiation Association Discussion 6 Dynamic SCNV patterns of ctDNA in three patients with acquired resistance. a–c The SCNV patterns at baseline, PR, SD, and PD in cases 22, 9, and 3, respectively. Orange, SCNV patterns at baseline (BL); green, SCNV patterns at partial response (PR); blue, SCNV patterns at stable disease (SD); red, SCNV patterns at progressive disease (PD). The Circos software package was used to plot genomic SCNVs Fig. 5 Dynamic changes in CNI scores of ctDNA and representative scan images. a–d Visualization of dynamic changes in CNI scores and representative scan images are presented for four patients (cases 26, 22, 18, and 21). PR partial response, SD stable disease, PD progressive disease. e The dynamic changes in CNI scores of ctDNA from four patients. CNI scores copy number instability score Fig. 5 Dynamic changes in CNI scores of ctDNA and representative scan images. a–d Visualization of dynamic changes in CNI scores and representative scan images are presented for four patients (cases 26, 22, 18, and 21). PR partial response, SD stable disease, PD progressive disease. e The dynamic changes in CNI scores of ctDNA from four patients. CNI scores copy number instability score Fig. 6 Dynamic SCNV patterns of ctDNA in three patients with acquired resistance. a–c The SCNV patterns at baseline, PR, SD, and PD in cases 22, 9, and 3, respectively. Orange, SCNV patterns at baseline (BL); green, SCNV patterns at partial response (PR); blue, SCNV patterns at stable disease (SD); red, SCNV patterns at progressive disease (PD). The Circos software package was used to plot genomic SCNVs Fig. 6 Dynamic SCNV patterns of ctDNA in three patients with acquired resistance. a–c The SCNV patterns at baseline, PR, SD, and PD in cases 22, 9, and 3, respectively. Orange, SCNV patterns at baseline (BL); green, SCNV patterns at partial response (PR); blue, SCNV patterns at stable disease (SD); red, SCNV patterns at progressive disease (PD). The Circos software package was used to plot genomic SCNVs scores, while 11 (65%) achieved increases of the HER2 copy number, at progressive disease (Fig. S5). For the ﬁrst time, we found that the efﬁcacy of CNI score to monitor therapeutic response of anti-HER2 treatment was not inferior to the changes of HER2 copy number. identiﬁcation and genomic subclones detection could identify the dynamic emergence of resistant subclones during therapy25,27. Ofﬁcial journal of the Cell Death Differentiation Association Discussion In addition, we also pre- sented the dynamic changes of CEA, CA125, CA19-9, and CA72-4 of 14 patients throughout the treatment with series information of serum biomarkers (Fig. S4). The CNI scores decreased in 9/13 patients at PR or SD, accom- panied by reduction levels of CEA, CA19-9, CA125, or CA72.4. These results derived from our relatively small sample are worth validating in future studies. Our previous research revealed a high concordance of HER2 ampliﬁcation between ctDNA and tumor tissues in 56 patients with AGC16. Furthermore, patients exhibited a decrease in the HER2 copy number when they beneﬁted from trastuzumab therapy, and achieved an increase at progressive disease. In the present study, among 17 patients with HER2-positive AGC who received anti- HER2 therapy, 13 (76%) achieved increases of the CNI The noninvasiveness of ctDNA has been used to detect the acquired resistance mutations selected by treatment of nonsmall cell lung cancer, melanoma, and metastatic HER2-positive GC7,23–25. In the dynamic process of clone evolution under selective pressures induced by treatment, our research showed that, as expected, the CNI scores Ofﬁcial journal of the Cell Death Differentiation Association Page 7 of 9 Chen et al. Cell Death and Disease (2019) 10:697 scores, while 11 (65%) achieved increases of the HER2 copy number, at progressive disease (Fig. S5). For the ﬁrst time, we found that the efﬁcacy of CNI score to monitor therapeutic response of anti-HER2 treatment was not inferior to the changes of HER2 copy number. The concept that spatiotemporal evolution of genomic clones is involved in drug resistance had been explored by l d 25 26 l l d D A b d identiﬁcation and genomic subclones detection could identify the dynamic emergence of resistant subclones during therapy25,27. Recently, the presence and size esti- mation of ibrutinib-resistant subclones at baseline in patients with chronic lymphocytic leukemia was demon- strated by droplet-based microﬂuidic technology and growth kinetic analyses26. In the present study, we found h h SC A f D A h d d Fig. 5 Dynamic changes in CNI scores of ctDNA and representative scan images. a–d Visualization of dynamic changes in CNI scores and representative scan images are presented for four patients (cases 26, 22, 18, and 21). PR partial response, SD stable disease, PD progressive disease. e The dynamic changes in CNI scores of ctDNA from four patients. CNI scores copy number instability score Fig. Plasma collection and ctDNA extraction CNI score ¼ X Pb i¼mb Zi j j; Whole blood from patients was collected in cell-free DNA BCT tubes (Streck Laboratories, USA), and then centrifuged at 1600g for 10 min at 4 °C to separate plasma from blood cells. The supernatant was transferred into a fresh tube and centrifuged at 16,000g for another 10 min at 4 °C. ctDNA was extracted from a 1000 μL aliquot of plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany), and genomic DNAs from peripheral blood cells were extracted using the RelaxGene Blood DNA System (Tiangen Biotech Co., Ltd., China). The quality of DNA was examined by quantitative polymerase chain reaction and the 2100 Bioanalyzer (Agilent, USA). All of the samples were stored at −80 °C for further use. where mb and pb are the bins ranked m% and p%, respectively, according to the Z value (m = 95, p = 99). ChromGo (Yikon Genomics Inc., Shanghai, China) software was used to automatically analyze sequencing data and report abnormalities of chromosomes. We used software package Circos (http://circos.ca/), ideal for visualizing genome DNA, to plot patients’ SCNV at the baseline, PR, SD, and PD stages. For the clustering heat- map, we used the R package dendextend (https://cran.r- project.org/web/packages/dendextend/vignettes/ introduction.html) for hierarchical clustering and the R package ComplexHeatmap (https://www.rdocumentation. org/packages/) to obtain the heatmap. Discussion Recently, the presence and size esti- mation of ibrutinib-resistant subclones at baseline in patients with chronic lymphocytic leukemia was demon- strated by droplet-based microﬂuidic technology and growth kinetic analyses26. In the present study, we found that the SCNA patterns of ctDNAs with acquired drug resistance were largely unchanged compared with The concept that spatiotemporal evolution of genomic clones is involved in drug resistance had been explored by several studies25,26. Multiregional and ctDNA-based next generation sequencing for intratumoral heterogeneity Page 8 of 9 Chen et al. Cell Death and Disease (2019) 10:697 baseline SCNV patterns, which suggested that multiple factors from different levels are involved in drug resistance. and the average length of each read was 100 bp. Over 91.92% of bases had a sequencing quality score ≥Q30 in LC WGS. The QC of sequence data was performed as described previously16. The adapters and low-quality bases were ﬁltered and trimmed from the raw data using Trimmomatic (version 0.35). High-quality reads were mapped to the reference genome (hg19) using BWA (version 0.7.12-r1039). Whole-genome sequencing Low-coverage whole-genome sequencing (LC WGS) based on ctDNA samples was performed to analyze SCNAs. More than 5 ng of the ctDNA was used to build the library, and the sequencing was performed on an Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). Each sample had about 5 million paired-end reads, Materials and methods Study design A cohort of 55 patients with histopathologically con- ﬁrmed AGC who received chemotherapy or targeted therapy at Peking University Cancer Hospital were included in this study. Totally, 100 plasma samples from 100 healthy people, 55 pretherapeutic plasmas (26 has paired blood cells) from 55 AGC patients and 75 dynamic plasmas from 26 AGC patients were collected. The clin- ical data of patients was obtained from their medical records and the clinical response after drug treatment was evaluated by computed tomography and categorized as complete response, PR, SD, or PD, according to the RECIST 1.1 criteria9. This study was approved by the Medical Ethics Committee of Peking University Cancer Hospital, and written informed consent was obtained from all of the patients for their samples to be used in the future. zi ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ log2 xi 2 r ; where xi is the relative copy number of each bin. The CNI score was calculated according to the formula, as descripted previously28 Ofﬁcial journal of the Cell Death Differentiation Association Conclusions We employed a low-coverage WGS to quantify the chromosomal instability of plasma ctDNA and revealed the dynamic changes induced by drug treatment in AGC. Our ﬁnding suggests that chromosomal instability of ctDNA could be used to predict and monitor therapeutic response in GC, although validation in a larger cohort will be necessary. Copy number analysis of ctDNA and CNI score calculation Unique mapped reads were extracted from the align- ment reads (BAM ﬁle). The whole reference genome was divided into non-overlapped observation windows (bins) with a size of 1000 kB. The read number and guanine- cytosine (GC) content were calculated in each bin. The bin read count was normalized based on the GC content and on a reference dataset to represent the relative copy number, which was reported accordingly16. we used R (version 3.0.0) to graph the relative copy number of each bin to visualize CNVs. The relative read number (RRN) of each bin was then segmented by circular binary seg- mentation (CBS) algorithms to merge bins with similar trends and calculate the ﬁnal copy number segments. Then, we calculated the Z value of each bin according to the formula Publisher’s note 18. Maleki, S. S. & Rocken, C. Chromosomal instability in gastric cancer biology. Neoplasia 19, 412–420 (2017). 18. Maleki, S. S. & Rocken, C. Chromosomal instability in gastric cancer biology. Neoplasia 19, 412–420 (2017). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 19. Penner-Goeke, S. et al. The temporal dynamics of chromosome instability in ovarian cancer cell lines and primary patient samples. PLoS Genet. 13, e1006707 (2017). Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-019-1907-4). Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-019-1907-4). 20. Schutz, E. et al. Chromosomal instability in cell-free DNA is a serum biomarker for prostate cancer. Clin. Chem. 61, 239–248 (2015). 20. Schutz, E. et al. Chromosomal instability in cell-free DNA is a serum biomarker for prostate cancer. Clin. Chem. 61, 239–248 (2015). Received: 6 February 2019 Revised: 19 August 2019 Accepted: 26 August 2019 21. Zhang, W. et al. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy. Nat. Commun. 7, 12619–12633 (2016). 21. Zhang, W. et al. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy. Nat. Commun. 7, 12619–12633 (2016). 22. Smeets, D. et al. Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy. Nat. Commun. 9, 4112–4127 (2018). 23. Oxnard, G. R. et al. Noninvasive detection of response and resistance in EGFR- mutant lung cancer using quantitative next-generation genotyping of cell- free plasma DNA. Clin. Cancer Res. 20, 1698–1705 (2014). Acknowledgements 14. Weiss, G. J. et al. Tumor cell-free DNA copy number instability predicts ther- apeutic response to immunotherapy. Clin. Cancer Res. 23, 5074–5081 (2017). 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Oellerich, M. et al. Using circulating cell-free DNA to monitor personalized cancer therapy. Crit. Rev. Clin. Lab. Sci. 54, 205–218 (2017). Conﬂict of interest The authors declare that they have no conﬂict of interest. 17. Wang, K. et al. Whole-genome sequencing and comprehensive molecular proﬁling identify new driver mutations in gastric cancer. Nat. Genet. 46, 573–582 (2014). 17. Wang, K. et al. Whole-genome sequencing and comprehensive molecular proﬁling identify new driver mutations in gastric cancer. Nat. Genet. 46, 573–582 (2014). 7. Wang, D. S. et al. Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer. Gut 68, 1152–1161 (2019). Ofﬁcial journal of the Cell Death Differentiation Association References MALBAC-based chromosomal imbalance analysis: a novel tech- nique enabling effective non-invasive diagnosis and monitoring of bladder cancer. BMC Cancer 18, 659–667 (2018). 28. Liu, H. et al. 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https://openalex.org/W1590458561	https://trialsjournal.biomedcentral.com/counter/pdf/10.1186/1745-6215-7-27	English	null	Protocol design and current status of CLIVIT: a randomized controlled multicenter relevance trial comparing clips versus ligatures in thyroid surgery	Trials	2,006	cc-by	4,752	Op Study protocol Protocol design and current status of CLIVIT: a randomized controlled multicenter relevance trial comparing clips versus ligatures in thyroid surgery CM Seiler1 3 BE Fröhlich3 JA Veit1 3 E Gazyakan3 MN Wente1 3 Address: 1Study Center of the German Surgical Society (SDGC), University of Heidelberg, Germany, 2Institute of Medical Biometrics and Informatics (IMBI), University of Heidelberg, Germany and 3Department of General-, Visceral-, Trauma Surgery, University of Heidelberg, Germany Email: CM Seiler - christoph_seiler@med.uni-heidelberg.de; BE Fröhlich - boris_froehlich@med.uni-heidelberg.de; JA Veit - johannes_veit@med.uni-heidelberg.de; E Gazyakan - gazyakan@bgu-ludwigshafen.de; MN Wente - moritz_wente@med.uni- heidelberg.de; C Wollermann - knauer@imbi.uni-heidelberg.de; A Deckert - deckert@imbi.uni-heidelberg.de; S Witte - witte@imbi.uni- heidelberg.de; N Victor - victor@imbi.uni-heidelberg.de; MW Buchler - markus_buechler@med.uni-heidelberg.de; HP Knaebel* - hanns- peter_knaebel@med.uni-heidelberg.de * Corresponding author Received: 14 July 2006 Accepted: 01 September 2006 Received: 14 July 2006 Accepted: 01 September 2006 Received: 14 July 2006 Accepted: 01 September 2006 Published: 01 September 2006 Trials 2006, 7:27 doi:10.1186/1745-6215-7-27 Trials 2006, 7:27 doi:10.1186/1745-6215-7-27 This article is available from: http://www.trialsjournal.com/content/7/1/27 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. Op Study protocol Protocol design and current status of CLIVIT: a randomized controlled multicenter relevance trial comparing clips versus ligatures in thyroid surgery CM Seiler1,3, BE Fröhlich3, JA Veit1,3, E Gazyakan3, MN Wente1,3, C Wollermann2, A Deckert2, S Witte1,2, N Victor2, MW Buchler1,3 and HP Knaebel1,3 Open Access BioMed Central BioMed Central BioMed Central Trials Trials Background ardized conditions is required in order to achieve high internal as well as external validity. This would allow the results to be generalized for thyroid surgery and also may have implications for other surgical settings. This study is one of the first studies designed and organized by the Study Center of the German Surgical Society (SDGC). Besides investigating the primary hypothesis, this study was also started with the aim of developing a nationwide structure for randomized trials using a common and sur- gical relevant procedure [14]. g Annually, a total of more than 90.000 surgical thyroid procedures are performed in surgical departments throughout Germany [1]. The prevalence of the endemic goiter in the adult population ranges between 20% and 30% in this iodine deficiency area [2]. Today, thyroid sur- gery is a highly standardized procedure with low morbid- ity and mortality. Several techniques of occluding vessels, besides the classic ligatures, are currently available. The clamp and tie technique is still the most frequently used standard, although it requires several steps which in addi- tion are time-consuming. An alternative technique is the application of vascular clips that have proven to be fast, effective and safe for achieving hemostasis in different sur- gical areas [3,4]. In thyroid surgery, however, no rand- omized controlled trial (RCT) has so far been performed comparing vascular clips with the conventional clamp and tie technique. Only ultrasonically activated shears were tested against the conventional technique in a RCT and found to be a suitable device in total thyroidectomies and lobectomies [5,6]. However, Voutilainen et al. (2000) stated that the advantage of the ultrasonically activated shears in thyroid surgery is probably of less importance for surgeons who achieve good results with vascular clips. This article describes the design and current status of the CLIVIT trial to publicly lay open the assumptions that have been made at the beginning of the trial. According to Chan et al. (2004) it is not uncommon that primary end- points are changed during the conduction of a clinical trial leading to substantial protocol violations and thereby misleading the medical community [15]. A protocol pub- lication might help to prevent this. Surgical trials are still inferior in their internal validity compared to pharmaco- logical studies [16]. Especially the clear description of concealed allocation sequences, interventions and assess- ment of surgeons' experience, safety issues and outcome evaluation are of interest. Need for this study To date, no clear scientific recommendation exists in regard to the available blood vessel ligation techniques in thyroid surgery. A systematic review comparing different blood vessel ligation techniques has not yet been per- formed. The literature on this topic shows several deficits thus reducing its internal and external validity. Main problems are underpowered sample sizes [6,13], missing data in regard to the surgical technique [5], lack of stand- ardization of the various surgical techniques [5] and of homogeneity of the used study designs [6]. Methods/design Trial population h l b l Besides achieving the best surgical outcome (low morbid- ity and mortality as well as absence of recurrence), operat- ing time is an important factor for thyroid surgeons [7- 10]). A reduction in operating time is not only beneficial for the patients, due to reduction of the risk associated with the time of general anaesthesia, but also for the health care system, because every minute of operating time saves approximately 8–11 € [6,11]. Hence, there is a large interest in performing surgery that is not only safe, but also fast and cost effective. Especially in centers with more than 60 – 100 thyroidectomies annually, the intro- duction of methods which potentially may reduce operat- ing time, will significantly increase the level of effectiveness [12]. The major eligibility criteria is a benign disease of the thy- roid gland – except Graves disease – scheduled for elective (a) hemithyroidectomy and subtotal resection of the other side, (b) near total or (c) total thyroidectomy. Malignant diseases of the thyroid gland, hyperparathy- roidism, re-operations and Graves disease were excluded from the study for they do have a higher risk for damage of the recurrent laryngeal nerve in surgery and/or they are not standard operations in smaller hospitals [17]. Detailed inclusion and exclusion criteria are specified in Table 1. Patients are screened consecutively for eligibility in all participating centers after approval of the study pro- tocol by the local ethics committee. A contract has been signed by the SDGC and the participating hospitals for correct conduction of the trial according to Good Clinical Practice. All participating surgeons performing the inter- ventions have been instructed by detailed manuals and the clips as well as their reusable applicators are provided. Abstract Background: Annually, more than 90000 surgical procedures of the thyroid gland are performed in Germany. Strategies aimed at reducing the duration of the surgical procedure are relevant to patients and the health care system especially in the context of reducing costs. However, new techniques for quick and safe hemostasis have to be tested in clinically relevance randomized controlled trials before a general recommendation can be given. The current standard for occlusion of blood vessels in thyroid surgery is ligatures. Vascular clips may be a safe alternative but have not been investigated in a large RCT. Methods/design: CLIVIT (Clips versus Ligatures in Thyroid Surgery) is an investigator initiated, multicenter, patient-blinded, two-group parallel relevance randomized controlled trial designed by the Study Center of the German Surgical Society. Patients scheduled for elective resection of at least two third of the gland for benign thyroid disease are eligible for participation. After surgical exploration patients are randomized intraoperatively into either the conventional ligature group, or into the clip group. The primary objective is to test for a relevant reduction in operating time (at least 15 min) when using the clip technique. Since April 2004, 121 of the totally required 420 patients were randomized in five centers. Discussion: As in all trials the different forms of bias have to be considered, and as in this case, a surgical trial, the role of surgical expertise plays a key role, and will be documented and analyzed separately. This is the first randomized controlled multicenter relevance trial to compare different vessel occlusion techniques in thyroid surgery with adequate power and other detailed information about the design as well as framework. If significant, the results might be generalized and may change the current surgical practice. Page 1 of 6 (page number not for citation purposes) (page number not for citation purposes) Trials 2006, 7:27 http://www.trialsjournal.com/content/7/1/27 http://www.trialsjournal.com/content/7/1/27 Page 2 of 6 (page number not for citation purposes) Objectives and outcomes The primary objective of this study is to test whether there is a relevant reduction in operating time (>15 min), using the clip vessel occluding technique versus the conven- tional clamp-tie. Secondary objectives are divided into surgical and non-surgical categories. In the surgical cate- gory there are the following points: total duration of oper- ation (skin incision until suture), weight of specimen, amount of postoperative bleeding, frequency of reopera- tion due to bleeding, recurrent laryngeal nerve paralysis (temporary or irreversible – pre- and postoperative assess- ment of vocal cord function by ENT-specialist), wound infection rate and rate of impaired parathyroid gland Interventions A standardized surgical approach to the thyroid gland will be performed in both groups: The skin is cut with a conventional scalpel (Kocher's inci- sion). Subcutaneous layer and platysma are cut with the electric cautery. After splitting cervical fascia, the infrahy- oideal muscles are set aside with retractors. Cervical mus- cles should be cut only exceptionally. The thyroid gland is then completely exposed in order to define the extent of the required surgical procedure. If the findings are in accordance with the trial inclusion criteria, randomiza- tion is performed. Regardless of the randomization result the procedure always starts with preparation and ligation of the upper pole vessels on the side of the hemithyroid- ectomy (Novafil® 3-0 or equivalent suture material). At the moment of dividing the upper pole vessels the time is taken and the operation then continues according to the regulations of the specified randomization group. Ligature Group In the ligature group each vessel will be occluded by man- ual ligature (Vicryl® 3-0 or equivalent suture material) using the square-knot technique and divided with scissors after ligation. INCLUSION CRITERIA • Malignant disease or highly suspicion for malignancy (clinical and imaging evidence) • Nerve palsy • Graves' Disease • Coagulopathy • Current immunosuppressive therapy • Chemotherapy within 2 weeks before operation • Radiotherapy completed no longer than 8 weeks before operation • Severe psychiatric or neurologic disease • Drug- and/or alcohol-abuse • Participation in another intervention-trial with interference of intervention and outcome • Inability to follow the instructions given by the investigator or the telephone interviewer • Lack of compliance At study enrollment: • Age equal or greater than 18 years • Expected survival time more than 12 months • Patients with benign diseases of the thyroid gland scheduled for elective surgery • Euthyroid metabolism (normal level of TSH or T3/T4) • Normal function of the vocal cords • Informed consent • Age equal or greater than 18 years • Normal function of the vocal cords • Informed consent At the end of surgical exploration: • Suitable for at least 2/3 resection of the thyroid At the end of surgical exploration: • Suitable for at least 2/3 resection of the thyroid At the end of surgical exploration: • Suitable for at least 2/3 resection of the thyroid For safety reasons concomitant medication and medical history are checked prior to operation. Patients at higher risk for complications (e.g. bleeding disorder, history of neck operation) are excluded from the study. All serious adverse events (SAE) will be reported to the principal investigator and the leading ethics committee. For the safety analysis the incidence of adverse events (AE) and SAE will be analyzed. Patients may withdraw from the study at any time either at their own request or at the request of the principal investigator. culap Challenger® clip applicator (BBD-Aesculap, Tuttlin- gen, Germany). Both groups The bipolar diathermy as an additional tool is allowed in both study groups. Time measurement stops as soon as the thyroid gland is removed. Following the resection, the capsule is sutured with an inverted continuous suture technique (PDS® 4-0 or equivalent suture material). After hemostasis uni- or bilateral drains (10 French sized Redons-drains) are placed into the resection area. Cervical muscles in the mid-line and platysma are adapted with conventional interrupted sutures. The operation is com- pleted with intracutaneous sutures and sterile wound dressing. Page 3 of 6 (page number not for citation purposes) Clinical sites and safety aspects Sites are selected according to their experience in thyroid surgery and willingness to adhere to the clinical trial pro- tocol. The surgical expertise of each participating surgeon is documented and taken into account during the analysis of the trial data. The participating centers are listed at the end of this paper. Therefore a randomized controlled multicenter, patient- blinded, two-group parallel relevance study under stand- Page 2 of 6 (page number not for citation purposes) http://www.trialsjournal.com/content/7/1/27 Trials 2006, 7:27 Table 1: CLIVIT trial: study inclusion and exclusion criteria Sample size A relevant difference between the two operations is defined as greater than 15 minutes. The expected differ- ence is 20 minutes. Analysis of retrospective data showed a standard deviation of about 27 minutes for the whole operation. For the primary endpoint a percentage of 70% of the whole operation time is assumed. If this percentage were exactly 70% for every operation, standard deviation for the primary endpoint would be 0.727 min = 18.9 minutes. Since this relation will probably not be so strict, the standard deviation for the primary endpoint is esti- mated to be higher. Thus, a standard deviation of 20 min- utes is assumed. The α level is set to 0.05; therefore, 200 patients per group are required to obtain a power of 80% (β = 0.2) using a simple one-sided t-test for two groups with a shifted null-hypothesis. Due to the fact that no interaction is expected regarding surgeon experience and treatment group, the sample size calculation is also valid for the ANOVA model of the primary analysis. In order to incorporate an expected early dropout rate of 5%, a total of 420 patients is required. A confirmatory analysis of variance (ANOVA) is planned for the primary endpoint based on the intention-to-treat (ITT) principle to test at first the hypothesis H0: μlig-μclip≤0 and in case of statistical significance the hypothesis H0: μlig-μclip≤15, with the expected operating times μlig and μclip, each on the α-level of 5%. The a priori ordered hypotheses hold the overall α-level of 5%. A randomized patient belongs to the ITT population after the upper ves- sels have been cut. The fixed factors of this ANOVA will be vessel occlusion technique, center and surgeons experi- ences. The relation between applied technique and dura- tion of procedure is severely influenced by the surgeon's expertise. Therefore experience is categorized into two groups: surgeons having performed ≥ 50 or <50 thyroid operations. Since there are so far no publications on this topic, the cut-off value of 50 operations was chosen, based on the results of an internal pilot study of the University of Heidelberg on learning curves of surgeons,. Even if neither superiority nor relevance can be shown sta- tistically, the exploratory interpretation of the narrow two-sided confidence intervals (expected width <10 min- utes) can be used to compare the difference in operating time. Randomization and blinding A block randomization list with alternating block sizes was generated by the Institute for Medical Biometrics and Informatics (IMBI) using SAS (SAS Version 8.2, SAS Insti- tute Inc., Cary, USA) for each center. The randomization code is stored at the IMBI and is not accessible to any clin- ical investigator. Until opening the sealed envelopes the allocation sequence is concealed in envelopes. If the indi- cation for a trial randomization is not given after explora- tion, the sealed envelope will be used for the next patient. Prerequisite for this is an intact seal on the opaque rand- omization envelope. Envelopes are used because any elec- tronic or telephone systems would have substantially increased the costs of the trial without any funding avail- able to cover these. Furthermore, easily accessible compu- ter systems are not available in all surgical theatres of the participating centers. Secondary endpoints will be analyzed and characterized using descriptive statistics. An interim analysis is not planned. Statistical analysis The participating centers collect the data and send the case report forms to the SDGC in Heidelberg. Double data entry is done and queries are generated for statistical mon- itoring. The analysis will be performed by the IMBI in cooperation with the SDGC using an analysis plan. Sample size Some sensitivity analyses are planned, using a linear mixed model as well as using the per-protocol population which consists of all patients of the ITT population with- out major protocol violations. http://www.trialsjournal.com/content/7/1/27 http://www.trialsjournal.com/content/7/1/27 function. The non-surgical category consists of duration of postoperative hospital stay. To assess the relevance of the chosen endpoints, the following ten aspects have to be ranked from "most important" to "least important" by patients and surgeons (only once): Intraoperative compli- cations, postoperative complications, length of hospital stay, voice function, dysphagia, death, postoperative pain, postoperative fatigue, convalescence of complete physical maximum resilience and cosmetic result [18]. After randomization the assigned group result is con- cealed. Subsequent investigations are performed by blinded study nurses or blinded investigators in order to assure unbiased assessment of endpoints when possible. Clip Group In the Clip Group each vessel will be occluded by applica- tion of one medium size clip close to the thyroid and two medium-size clips distal to the thyroid using the BBD-Aes- Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) Trials 2006, 7:27 http://www.trialsjournal.com/content/7/1/27 Page 4 of 6 (page number not for citation purposes) http://www.trialsjournal.com/content/7/1/27 http://www.trialsjournal.com/content/7/1/27 investigators from the SDGC to ensure correct patient recruitment and meticulous documentation. investigators from the SDGC to ensure correct patient recruitment and meticulous documentation. mittee gave a positive vote on January 7th, 2004. All participants will be informed about the trial and the writ- ten informed consent must be signed before randomiza- tion. The trial was registered in December 2003 at the International Standard Randomized Controlled Trial Number Registration (ISRCTN96901396). Randomization bias. Randomization is done in a block randomization system with alternating block sizes to avoid positive prediction of the last procedures in each block. The calculated randomization sequence is sealed at the IMBI for each participating center. Envelopes are non- transparent and cannot be resealed after opening. After exploration of the thyroid gland and affirmation of the indication an intra-operative randomization is per- formed. The unclosed randomization sheet is sent via fax to the IMBI. Two Amendments were accepted before this publication by the ethical committees: in the first amendment two additional secondary objectives (total time of the proce- dure, weight of specimen) were added. This was done in order to improve evaluation of the primary objective 'resection time' which is calculated by measuring the time between the completion of ligation of the upper pole ves- sels and the time of removal of the complete specimen. The second amendment was introduced to expand the inclusion criteria in regard to the type of operation. Until then, only hemi-thyroidectomies in combination with subtotal thyroid resection of the other side were included into the trial. Since this operation is not the only standard surgical procedure in goiter surgery in Germany, complete thyroidectomy and near-total thyroidectomy were added. The surgical details of these techniques are not in conflict with the objectives of the study. By this second amend- ment a shorter recruitment period is expected. Surgeon's expertise bias. Another source of bias is the sur- geon's expertise. Most often new surgical techniques or methods are introduced by the most experienced sur- geons. To minimize this source of bias stratification for surgeon's skill is made, and measured data will be ana- lyzed by analysis of variance in regard to surgeon's skill, center and vessel occlusion technique. Due to this analysis of variance, bias of center effects and surgeon's skill should be reduced. Current status and duration of the trial Up to now six centers are initiated and 155 patients, of the 400 needed, were randomized by five centers (status July 5th, 2006, details see acknowledgements). The first patient was randomized on March 5th, 2004. The expected recruit- ment period will last until the end of 2008. Prevention of non-conclusive results. Recent reports com- paring different methods of hemostasis in thyroidecto- mies were underpowered or not designed as RCTs. In the calculation of the sample size of 420 patients, a post ran- domization drop out of 10 patients in each allocation arm and additional 40 patients for each secondary endpoint for 'lost to follow up' are included. Thus, the calculated sample size is large enough to draw definite conclusions. The interpretation of the hypothesis has a high statistical power: a significant result of this relevance test is equiva- lent to a time difference of at least 15 minutes [19]. http://www.trialsjournal.com/content/7/1/27 Possible complications such as bleed- ing, re-operation due to bleeding, recurrent palsy or impaired function of parathyroid glands are assessed by blinded study nurses or investigators. Re-operation, recur- rent palsy and impaired function of parathyroid glands are obvious events that can not be diminished by the responsible surgeon. Trial organization, quality control, registration and ethical aspects The trial was designed at the Study Center of the German Surgical Society (Studienzentrum der Deutschen Gesells- chaft für Chirurgie, SDGC) in cooperation with the Insti- tute of Medical Biometrics and Informatics (IMBI) at the University of Heidelberg, Medical School. Trial monitor- ing is done by an independent monitor of the SDGC. The trial is performed according to the Declaration of Helsinki in its current German version and the Good Clinical Prac- tice (GCP). Before trial start the independent ethic com- Randomization and time measurement is performed by an independent member of the clinical site (study nurse or anesthesiologist) who is not involved in the surgical procedure. Page 4 of 6 (page number not for citation purposes) Page 4 of 6 (page number not for citation purposes) Trials 2006, 7:27 http://www.trialsjournal.com/content/7/1/27 Trials 2006, 7:27 Discussion The evaluation of optimal surgical therapeutic options and procedures should be performed according to the best current knowledge. Due to the principles of Evidence- based Medicine (EbM), the randomized controlled trial (RCT) is assumed to be the gold standard in evaluating different medical methods and procedures. Even in RCTs, though, the risk of bias should not be underestimated. In surgical trials a double blinded trial design is difficult to establish; therefore, the patient selection, power, rand- omization, surgeon's skill, data assessment and documen- tation of outcome measures and statistical analyses are important design issues and susceptible to the different forms of bias. Hence, all these issues are specified in the trial protocol a priori. Publication bias. One important tool to avoid bias in clin- ical trials is the international registration and publication of trial protocols. CLIVIT was registered in 2003 before the subsequent requirement of the International Committee of Medical Journal Editors were published [20]. In conclusion, this is the first randomized controlled mul- ticenter relevance trial comparing different vessel occlu- sion techniques in thyroid surgery with adequate power and sufficient detailed information about the design as well as framework. If significant, the results might be gen- eralized and may change the current surgical practice. Selection bias. To minimize the selection bias all patients scheduled for thyroid surgery are screened consecutively in all participation sites. Moreover, neither patient age nor the size of the goiter is an exclusion criterion. Screening lists and case report files are monitored by independent Page 5 of 6 (page number not for citation purposes) Page 5 of 6 (page number not for citation purposes) http://www.trialsjournal.com/content/7/1/27 Trials 2006, 7:27 http://www.trialsjournal.com/content/7/1/27 analysis and evaluation of the clinical relevance of study var- iables by the patient and physician]. Chirurg 2001, 72:19-28. Victor N: On clinically relevant differences and shifted null hypotheses. Methods Inf Med 1987, 26:109-116. De Angelis C, Drazen JM, Frizelle FA, Haug C, Hoey J, Horton R, Kotzin S, Laine C, Marusic A, Overbeke AJ, Schroeder TV, Sox HC, Van Der Weyden MB: Clinical trial registration: a statement from the International Committee of Medical Journal Edi- tors. Lancet 2004, 364:911-912. Competing interests p g The author(s) declare that they have no competing inter- ests. 19. 20. References 1. 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Bergamaschi R, Becouarn G, Ronceray J, Arnaud JP: Morbidity of thyroid surgery. Am J Surg 1998, 176:71-75. y g y J g , 8. Roher HD, Goretzki PE, Hellmann P, Witte J: [Complications in thyroid surgery. Incidence and therapy]. Chirurg 1999, 70:999-1010. 9. Thomusch O, Machens A, Sekulla C, Ukkat J, Lippert H, Gastinger I, Dralle H: Multivariate analysis of risk factors for postopera- tive complications in benign goiter surgery: prospective mul- ticenter study in Germany. World J Surg 2000, 24:1335-1341. 10. Thomusch O, Machens A, Sekulla C, Ukkat J, Brauckhoff M, Dralle H: The impact of surgical technique on postoperative hypopar- athyroidism in bilateral thyroid surgery: a multivariate anal- ysis of 5846 consecutive patients. Surgery 2003, 133:180-185. 11. Koperna T: How long do we need teaching in the operating room? The true costs of achieving surgical routine. Langen- becks Arch Surg 2004, 389:204-208. g 12. Friess H, Kleeff J, Buchler P, Hartwig W, Schmidt J, Radnic S, Auer S, Buchler MW: [Central patient management in surgery]. Chirurg 2002, 73:111-117. g 13. Siperstein AE, Berber E, Morkoyun E: The use of the harmonic scalpel vs conventional knot tying for vessel ligation in thy- roid surgery. Arch Surg 2002, 137:137-142. g y g 14. Knaebel HP, Diener MK, Wente MN, Bauer H, Buchler MW, Roth- mund M, Seiler CM: The Study Centre of the German Surgical Society-rationale and current status. Langenbecks Arch Surg 2005, 390:171-177. Acknowledgements g The trial is designed, managed and conducted by the Study Center of the German Surgical Society (SDGC. It is funded by a grant from the German Ministry of Research and Education and supported by the BBD-Aesculap© Company, Tuttlingen, Germany. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 15. Chan AW, Hrobjartsson A, Haahr MT, Gotzsche PC, Altman DG: Empirical evidence for selective reporting of outcomes in randomized trials: comparison of protocols to published articles. JAMA 2004, 291:2457-2465. J 16. Boutron I, Tubach F, Giraudeau B, Ravaud P: Methodological dif- ferences in clinical trials evaluating nonpharmacological and pharmacological treatments of hip and knee osteoarthritis. JAMA 2003, 290:1062-1070. J 17. Tresallet C, Chigot JP, Menegaux F: [How to prevent recurrent nerve palsy during thyroid surgery?]. 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https://openalex.org/W2913333940	https://www.nature.com/articles/s41598-018-36905-z.pdf	English	null	3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain	Scientific reports	2,019	cc-by	9,065	3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain P. Joy Dunmore-Buyze 1, Charmainne Cruje1,2, Zengxuan Nong1, Jason J. Lee1,2, f y P. Joy Dunmore-Buyze 1, Charmainne Cruje1,2, Zengxuan Nong1, Jason J. Lee1,2, John A. Kiernan 3, J. Geoffrey Pickering 1,2,4,5 & Maria Drangova 1,2 Received: 3 September 2018 Accepted: 23 November 2018 Published: xx xx xxxx Received: 3 September 2018 Accepted: 23 November 2018 Published: xx xx xxxx Received: 3 September 2018 Accepted: 23 November 2018 Published: xx xx xxxx Virtual histology – utilizing high-resolution three-dimensional imaging – is becoming readily available. Micro-computed tomography (micro-CT) is widely available and is often coupled with x-ray attenuating histological stains that mark specific tissue components for 3D virtual histology. In this study we describe a new tri-element x-ray attenuating stain and perfusion protocol that provides micro-CT contrast of the entire vasculature of an intact mouse. The stain – derived from an established histology stain (Verhoeff’s) – is modified to enable perfusion through the vasculature; the attenuating elements of the stain are iodine, aluminum, and iron. After a 30-minute perfusion through the vasculature (10-minute flushing with detergent-containing saline followed by 15-minute perfusion with the stain and a final 5-minute saline flush), animals are scanned using micro-CT. We demonstrate that the new staining protocol enables sharp delineation of the vessel walls in three dimensions over the whole body; corresponding histological analysis verified that the CT stain is localized primarily in the endothelial cells and media of large arteries and the endothelium of smaller vessels, such as the coronaries. The rapid perfusion and scanning protocol ensured that all tissues are available for further analysis via higher resolution CT of smaller sections or traditional histological sectioning. Advances in vascular research rely heavily on detailed characterization of rodent models of disease. Histology is commonly employed to investigate the architecture and constituents of the vasculature and organs of mice1–3 and study various disease models4–7. Histological protocols require the excision of tissue followed by fixation, dehydration, clearing and embedding, cutting into thin sections (5–10 μm) and immersing in chemical stains for varying periods of time. Although standard histology provides images with high resolution, it is limited by the requirement for processing a limited number of sections, thereby losing three-dimensional context. Furthermore, traditional histological staining protocols are time-consuming and are prone to introducing morphological arti- facts8. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 3 September 2018 Accepted: 23 November 2018 Published: xx xx xxxx Materials and Methods Animal handling and preparation. Animal studies were performed under a protocol approved by the University Council on Animal Care at the University of Western Ontario, which follows the policies set out by the Canadian Council on Animal Care. The “CT stain” perfusion techniques presented in the paper were developed using 10 mice (C56BL/6 male, 25–30 g, Jackson Laboratory, Bar Harbor ME) for each stain studied; results are reported from two mice for each of the CT stains. Prior to cannulation of the abdominal aorta, mice were anaes- thetized with an intraperitoneal injection of 50 mg/kg of ketamine hydrochloride and 12.5 mg/kg of xylazine; to prevent clotting during the perfusion, 0.2 mL of heparin (Sodium Injection USP, 200 units, Sandoz Canada Inc.) was injected intraperitoneally. A midline incision was made in the abdomen, beginning just below the diaphragm. The abdominal aorta was dissected free and cannulated using a 24 GA, 19 mm long I.V. catheter; the catheter was attached to the perfusion system (see below) and the inferior vena cava was cut to allow drainage of the perfusate. CT stains. Three different CT stains were evaluated. All solutions were filtered prior to perfusion and were used within 24 h of preparation in de-ionized water. Following prior work24 5% w/v PTA and I2KI (2.5% w/v KI and 1.3% w/v I2) solutions were prepared. A modified Verhoeff’s solution was also prepared, where the modification consisted of replacing the traditional alcoholic heamatoxylin in the formulation with an aluminum-heamatoxylin solution (Surgipath, Harris Hematoxylin, Leica Biosystems). The stain components were modified because the traditional Verhoeff’s stain33 (containing 5% w/v heamatoxylin in ethanol) caused precipitation when perfused through the animal and prevented complete perfusion by occluding small vessels. Specifically, the modified solution contained 56% v/v aluminum (Harris) heamatoxylin, 2.2% w/v FeCl3•6H2O, 0.6% w/v KI and 0.3% w/v I2. Perfusion system. Consistency of the perfusions was facilitated by the use of a modified ValveBank®8 Pinch Valve (Automate Scientific, Berkeley, CA) gravity-fed perfusion system, which seamlessly switches between solu- tions (Supplementary Fig. S1). The pinch valve apparatus of the commercial system was redesigned to allow for the use of larger diameter tubing (4.76 mm), which provided the flow rates and pressure required to perfuse whole mice and rats. Up to 6 different solutions can to be perfused, as required, without moving the animal once the catheter is in place. Saline flush with and without a detergent. www.nature.com/scientificreports/ tissues. Our group has developed a whole-body perfusion technique, which obviates the need to extract and soak individual organs or tissues. This technique exploits the vasculature as a channel to deliver stains throughout the entire body24 tissues. Our group has developed a whole-body perfusion technique, which obviates the need to extract and soak individual organs or tissues. This technique exploits the vasculature as a channel to deliver stains throughout the entire body24.h y The previously studied stains are of interest in micro-CT imaging because they contain heavy elements (i.e. iodine, tungsten and osmium). Since these stains have known affinities for specific tissue components – such as PTA for collagen25–27, fibrin3, muscle fascicles16, and extracellular tissue proteins in the glomeruli9 or I2KI for soft tissues such as myocardial muscle fibres28 and various connective tissues17,18,29 – their presence in micro-CT images would coincide with the abundance of the tissue component it selectively stains. For models of vascular disease, stains that bind to endothelial cells, collagen, extracellular matrix proteins and elastin would be advanta- geous. While I2KI, PTA, and OsO4 are useful in studying the vessel wall as a whole unit, no micro-CT stain that highlights individual wall components has been demonstrated. A commonly used histology stain for elastic tissue and cell nuclei is Verhoeff’s stain, which is reported to bind to elastin by van der Waals forces30–32. Verhoeff’s stain contains two x-ray attenuating materials (iron and iodine), making it an attractive candidate as a micro-CT stain for vascular analysis. y In this study, we describe a novel, tri-element x-ray attenuating stain and perfusion protocol derived from Verhoeff’s stain that provides micro-CT contrast of the entire vasculature of an intact mouse. Rapid perfusion – of less than 30 minutes – with the novel stain results in micro-CT images of vessel walls, which compared favourably with images of I2KI- and PTA-perfused mice. Corresponding histological sections localized the heavy elements within the vessel wall. We describe our novel stain and demonstrate its ability to provide 3D images of vessel walls throughout the entire mouse vasculature, and hence its utility in virtual histology; to our knowledge, no other CT staining technique localizes contrast within the structural components of the vessel wall, thereby producing whole-mouse vasculature-specific images using 3D micro-CT, following a perfusion procedure under 30 minutes. 3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain P. Joy Dunmore-Buyze 1, Charmainne Cruje1,2, Zengxuan Nong1, Jason J. Lee1,2, f A technique that enables the microanatomy of rodents to be studied in three dimensions (3D) while leaving organs of interest intact would allow for structural and morphological information to be obtained9–13.f g g Micro-computed tomography (micro-CT) is a cost-effective and widely available 3D imaging technique that provides an opportunity to non-destructively study intact rodents and explanted tissues at high resolution. Recently, a strategy of using histological stains that mark specific tissue components combined with micro-CT has been applied in “virtual histology” – a technique that can be used to study the microstructure of tissues without disturbing the morphology of an organ or tissue of interest. Several groups have used high resolution micro-CT for virtual histology to visualize soaked excised organs and tissues in iodine-potassium iodide (I2KI)14– 20, phosphotungstic acid (PTA)8,9,21, and osmium tetroxide (OsO4)22 for periods of time ranging from hours to weeks. Previous reports have also shown that unstained tissue can be visualized using other x-ray based imaging techniques, such as phase contrast CT8 and photon counting23. However, these techniques have limited field of view and require the removal of tissue from its native anatomy. Since the vascular system is a network that encompasses the whole body, there is great value in observing the gross vasculature in addition to imaging select 1Robarts Research Institute, The University of Western Ontario, London, Ontario, N6A 5B7, Canada. 2Department of Medical Biophysics, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. 3Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. 4Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. 5Departments of Medicine and Biochemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada. P. Joy Dunmore-Buyze and Charmainne Cruje contributed equally. Correspondence and requests for materials should be addressed to M.D. (email: mdrangova@robarts.ca) Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 1 www.nature.com/scientificreports/ Materials and Methods The image slices shown were selected to demonstrate uptake by the aorta (arrowheads), heart (circle), and the liver (arrow) in the top row, and kidneys (asterisk) in the bottom row, which are labeled in (e) for reference. with 50 µm isotropic voxels and intensities were converted to Hounsfield units (HU), where the image intensities scale linearly with attenuation and the value of water equals 0 HU and that of air equals −1000 HU. Following micro-CT scanning, tissue samples were harvested for histology. Image visualization and analysis. The micro-CT images were viewed and analysed using MicroView (Parallax Innovations, London, ON). While the general “binding affinity” of the CT stains is consistently repro- ducible, absolute increases in x-ray attenuation is variable between specimens and tissue types. Nevertheless, quantitative evaluation of the attenuation (in HU) of representative tissues is valuable27 and was performed for two mice perfused with each CT stain. Because the murine vessel wall is less than 100 µm thick, regions of interest were defined as 0.5-mm (10-voxel) long single-voxel line profiles drawn along the length of the ascending aorta in three consecutive slices (all stains) and along select coronary arteries (AlumHemFeI and AlumHemFeI-T only); for each region the mean and standard deviation were calculated. (In essence, the regions of interest represent 3 × 10 × 1 voxel patches of the vessel wall.) For reference, similar regions of interest were defined in the humerus (forelimb), which is representative of cortical bone. Because the entire vasculature is perfused during the exper- iment, unstained soft tissue cannot be identified consistently in the mice and therefore no attempt was made to calculate contrast enhancement over background. Lastly, for comparison to prior work24, 0.5 × 0.5 × 0.5 mm regions of interest were used to measure attenuation in the myocardium of the PTA- and I2KI-stained mice. The measured values were averaged to generate a mean CT number for each tissue type. The mean of the standard deviations of the regions of interest from the two mice are also reported; these standard deviations represent a combination of image noise and variability in regional stain delivery. Histological analysis. In order to identify the vessel-wall components to which the various chem- ical agents may be adhering to, histological processing tailored to each stain compound was performed. Materials and Methods Clearing of the blood prior to perfusion with a stain was achieved by perfusing the vasculature with heparinized saline (0.9% NaCl; 0.1% heparin). To increase the perme- ability of the vessel wall, a second solution was prepared by adding a non-ionic detergent (0.03% Triton™ X-100, BDH Analytical Chemicals) in phosphate buffered saline. CT-stain perfusion protocol. Mice were perfused via retrograde perfusion of the abdominal aorta at phys- iological pressure (~110 mm Hg). Heparinized saline – with or without Triton™ X-100 – was flushed through the vasculature for 10 minutes, which caused animal death by exsanguination. One of the three CT stains was then delivered using the following timing: 15 minutes for I2KI, 30 minutes for PTA, and 10 minutes for the aluminum-modified stain. These times were based on earlier work and preliminary tests aimed at optimizing vessel contrast (aluminum-modified CT stain). Finally, the vasculature was flushed with normal saline for 5 min- utes. We will refer to the aluminum-modified stain applied following saline flush as AlumHemFeI and to the stain applied following flushing with saline with Triton™ X-100 as AlumHemFeI-T. Additional mice were perfused with heparinized saline – with or without Triton™ X-100 – for 15 minutes to act as negative controls. Micro-CT imaging. Immediately following perfusion, whole body micro-CT scans were obtained on a GE eXplore speCZT scanner (GE Healthcare, London, ON) using a 5-minute protocol (900 views, 0.4° angle incre- ment). Scan parameters were 90 kVp, 40 mA, 2 × 2 binning, and 16 ms exposure time. Images were reconstructed Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 2 www.nature.com/scientificreports/ Figure 1. Coronal slices from micro-CT volume images (50 µm isotropic voxels) of whole-body perfused mice. The images illustrate the difference in contrast enhancement based on the stain perfused: (a) saline, (b) PTA, (c) I2KI, (d) AlumHemFeI and (e) AlumHemFeI-T. The image slices shown were selected to demonstrate uptake by the aorta (arrowheads), heart (circle), and the liver (arrow) in the top row, and kidneys (asterisk) in the bottom row, which are labeled in (e) for reference. Figure 1. Coronal slices from micro-CT volume images (50 µm isotropic voxels) of whole-body perfused mice. The images illustrate the difference in contrast enhancement based on the stain perfused: (a) saline, (b) PTA, (c) I2KI, (d) AlumHemFeI and (e) AlumHemFeI-T. Materials and Methods The I2KI-perfused tissues were removed from the perfused animal and embedded in OCT so that frozen sections could be obtained; these frozen sections were examined with no additional processing or staining because I2KI appears brown in the sections and is largely removed by water or organic solvents. Samples from the PTA, AlumHemFeI and AlumHemFeI-T-perfused animals were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned (5 µm thick). Toluidine blue34, which combines with PTA to form an insoluble pig- ment, was used to counter-stain the colourless PTA-perfused samples. The iron-containing AlumHemFeI- and AlumHemFeI-T-stained sections were counter-stained with a Perls’ Prussian blue method35, to localize iron in the tissues. Sections from saline-perfused mice were also stained, as negative controls for the CT stains. Sections were viewed and photographed using an Olympus BX51 (Center Valley, PA) microscope. Results Q lit t Qualitative comparison of CT stains. Whole-body retrograde perfusion of CT stains was successfully – and reproducibly – achieved using the apparatus and protocols developed. The images of Fig. 1 confirm prior results, using hand perfusion, with PTA and I2KI. PTA weakly bound to the myocardium and enhanced the parenchymal structure of the liver (Fig. 1b). In contrast, I2KI clearly enhanced the myocardium and enabled visualization of myocardial structure, while the coronary artery lumina appear as voids (Fig. 1c). I2KI also bound to the walls of the aorta and liver vessels, but it diffused into the tissue surrounding the vessels. The AlumHemFeI stain, when perfused following saline flushing, demonstrated a great affinity for arterial wall, particularly that of the coronaries, renal arteries, aorta, and carotids. However, the stain also appeared to diffuse through the wall and enhance surrounding tissues, such as the myocardium and renal cortex (Fig. 1d). The inclusion of Triton™ Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Thick maximum intensity projections (1 mm thick) of the thorax and abdomen of mice perfused with the 4 different stains: PTA (a), I2KI (b), AlumHemFeI (c), and AlumHemFeI-T (d). It can be seen that the AlumHemFeI-T stain provides a clear delineation of arterial walls; the arrow points to the aortic arch. Figure 2. Thick maximum intensity projections (1 mm thick) of the thorax and abdomen of mice perfused with the 4 different stains: PTA (a), I2KI (b), AlumHemFeI (c), and AlumHemFeI-T (d). It can be seen that the AlumHemFeI-T stain provides a clear delineation of arterial walls; the arrow points to the aortic arch. X-100 in the saline flush, results in a clearer visualization of the arterial wall of all major organs with minimal diffusion into surrounding tissue (Fig. 1e). When the four staining protocols are compared, it is evident that the AlumHemFeI and AlumHemFeI-T provide delineation of the aortic wall better than that seen with PTA, compa- rable to that seen with I2KI, and providing, in addition, images of coronary, renal, and hepatic vessel walls.hf The effectiveness of AlumHemFeI-T in delineating the vessel wall is further demonstrated by observing the thick maximum intensity projections (MIPs, 1-mm thick) in Fig. 2. Results Q lit t Whereas PTA and I2KI stains marked whole organs, AlumHemFeI-T displayed a clear delineation of the vasculature feeding and within these organs.f g p y g g Figure 3 shows three different thick-slab MIPs from a single mouse and provides further convincing demon- stration that delivery of the AlumHemFeI-T solution resulted in concentrated and extensive staining of the vessel walls, without diffusing into the surrounding tissues. Figure 3a shows the aortic arch, coronary arteries including intramyocardial branches, and the liver vasculature. The AlumHemFeI-T stain can also be clearly seen in the common carotid arteries, the internal and external carotids, the kidney vasculature (Fig. 3b), and the small lum- bar vessels in the spine as well as the cerebral vessels (Fig. 3c). The oblique MIPs of Fig. 3 and the Supplementary Videos SV1–SV3 further demonstrate the ability to follow the arterial vasculature throughout the body. Quantitative analysis. The results of the quantitative analysis of CT enhancement provided by the four different staining protocols are presented in Table 1. These results show that each stain binds differently depend- ing on the tissue type. PTA generally provided weaker enhancement than the iodine-containing stains. While I2KI delivery yielded the highest CT numbers in the aortic wall, it did not delineate the coronary artery wall. Instead, coronary artery wall enhancement was quantifiable only in mice perfused with the AlumHemFeI and AlumHemFeI-T agents. Histology of CT-stained tissues. To relate the micro-CT images to histology, sections of excised aortas and hearts from perfused mice were examined. Light microscopy images of representative histological sections of heavy element stain-perfused mice are shown in Figs 4 and 5 for the aorta and myocardium/coronary arteries, respectively. Negative-control histological sections are of aorta and myocardium are provided in Supplementary Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Coronal (a,b) and sagittal (c) slices of an AlumHemFeI-T whole-body perfused mouse. Slices are taken from images acquired with a 5-minute scan protocol (90 kVp; 50 μm voxels). The images are 1-mm maximum intensity projections selected to highlight the coronary vasculature (circle in a), aortic arch (in a,b), common carotid arteries (arrows in a,b), the internal and external carotid arteries (arrowheads in b), the cerebral vasculature (arrow in c), and the small lumbar vessels (arrowheads in c). All images can bee seen by scrolling through the Supplementary Videos SV1, SV2 and SV3. Figure 3. Results Q lit t Coronal (a,b) and sagittal (c) slices of an AlumHemFeI-T whole-body perfused mouse. Slices are taken from images acquired with a 5-minute scan protocol (90 kVp; 50 μm voxels). The images are 1-mm maximum intensity projections selected to highlight the coronary vasculature (circle in a), aortic arch (in a,b), common carotid arteries (arrows in a,b), the internal and external carotid arteries (arrowheads in b), the cerebral vasculature (arrow in c), and the small lumbar vessels (arrowheads in c). All images can bee seen by scrolling through the Supplementary Videos SV1, SV2 and SV3. Tissue type CT stain PTA I2KI AlumHemFeI AlumHemFeI-T Saline mean* SD£ mean SD mean SD mean SD mean SD aorta 424 50 1623 72 913 30 731 23 — coronary 3063§ 176 1377§ 116 — myocardium 583 153 3338 124 101 37 cortical bone† 1968 71 1846 86 1690 151 1390 125 1452 93 Table 1. Quantitative contrast of various tissue types; all values are in HU. Mean of the regions of interest from two mice for each tissue type. £Mean of the standard deviations of the regions of interest from the two mice, representing noise and variability in regional stain delivery. §Higher attenuation observed in the coronary vessel walls of AlumHemFeI perfused mice compared to the AlumHemFeI-T mice is attributed to the increased localization of the stain within the vessel wall when AlumHemFeI-T is used. †Cortical bone values agree with prior data39; variations can be attributed to differences in age. _In the saline-perfused mice all non-fatty soft tissue has the same mean attenuation; no attempt was made to identify aorta or coronaries. Tissue type CT stain PTA I2KI AlumHemFeI AlumHemFeI-T Saline mean SD£ mean SD mean SD mean SD mean SD aorta 424 50 1623 72 913 30 731 23 — coronary 3063§ 176 1377§ 116 — myocardium 583 153 3338 124 101 37 cortical bone† 1968 71 1846 86 1690 151 1390 125 1452 93 Table 1. Quantitative contrast of various tissue types; all values are in HU. *Mean of the regions of interest from two mice for each tissue type. £Mean of the standard deviations of the regions of interest from the two mice, representing noise and variability in regional stain delivery. Results Q lit t §Higher attenuation observed in the coronary vessel walls of AlumHemFeI perfused mice compared to the AlumHemFeI-T mice is attributed to the increased localization of the stain within the vessel wall when AlumHemFeI-T is used. †Cortical bone values agree with prior data39; variations can be attributed to differences in age. _In the saline-perfused mice all non-fatty soft tissue has the same mean attenuation; no attempt was made to identify aorta or coronaries. Figs S2 and S3, respectively; these figures contain histological images of all CT stains evaluated. In each case, the negative-control sections appear colourless, verifying that any pigmentation in the CT stain-perfused tissues was due to the presence of the CT stain. In PTA-perfused sections, subsequent staining with toluidine blue marked the presence of PTA through- out the aortic wall, including the endothelium, the medial interlamellar units (between the elastic laminae) and adventitia (Fig. 4a). Within the myocardium (Fig. 5a) high intensity PTA-detection staining was observed in both the coronary vessel walls and the surrounding myocardial tissue. This observation is consistent with the micro-CT image (Fig. 2a) and indicates the delivery of the PTA stain into the myocardium, via the coronary vessels and capillaries.h In I2KI-perfused mice, iodine stains tissues brown. The brown colour was uniformly distributed across the wall of the aorta (Fig. 4b) and high-intensity colour is seen throughout the myocardium (Fig. 5b), while no coro- nary arteries were detectable. (Note, the poor quality of the micrographs is because, to avoid washing away of the tissue-associated iodine, frozen tissue sections maintained in OCT embedding media needed to be studied and Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 5 www.nature.com/scientificreports/ Figure 4. Sections of aortas from mice perfused with PTA (a), I2KI (b), AlumHemFeI (c) and AlumHemFeI-T (d). The images in (e) and (f) are magnifications of the regions outlined in c, and d respectively. The purple colour in (a) indicates the presence of PTA within the aortic wall. The iodine within the aortic wall is orange- brown in colour (frozen section). Both AlumHemFeI (c,e) and AlumHemFeI –T (d,f) stain the interlamellar spaces, but AlumHemFeI-T provides more intense staining of the endothelium (arrow) and the endothelial cell nuclei (arrowheads in f). For negative controls see Supplementary Fig. S2. Figure 4. Sections of aortas from mice perfused with PTA (a), I2KI (b), AlumHemFeI (c) and AlumHemFeI-T (d). imaged. Nonetheless, Figs 4b and 5b confirm that the CT enhancement seen in Fig. 2b is due to I2KI accumulation within the entire aortic wall and the myocardium. Figures 4c,d and 5c,d suggest that, interestingly, AlumHemFeI and AlumHemFeI-T stain specific compo- nents of the vessel wall. The Prussian blue reaction product showed the distribution of iron (in the aluminum, iodine, and iron heavy element mixture) as different intensities of blue. For AlumHemFeI, iron was present within the interlamellar units of the aorta (Fig. 4c,e and Supplementary Fig. S2), with the highest concentra- tion in the endothelium (Fig. 4e). Particularly noteworthy was the finding of intense blue also seen within some endothelial-cell nuclei (Fig. 4e and Supplementary Fig. S2). Within the heart (Fig. 5c,e and Supplementary Results Q lit t The images in (e) and (f) are magnifications of the regions outlined in c, and d respectively. The purple colour in (a) indicates the presence of PTA within the aortic wall. The iodine within the aortic wall is orange- brown in colour (frozen section). Both AlumHemFeI (c,e) and AlumHemFeI –T (d,f) stain the interlamellar spaces, but AlumHemFeI-T provides more intense staining of the endothelium (arrow) and the endothelial cell nuclei (arrowheads in f). For negative controls see Supplementary Fig. S2. Figure 5. Sections of myocardium from mice perfused with PTA (a), I2KI (b), AlumHemFeI (c) and AlumHemFeI-T (d). The images in (e) and (f) are magnifications of the regions outlined in c, and d respectively. The purple colour in (a) indicates the presence of PTA within the myocardium and coronary wall. Intense iodine staining of the myocardium is seen in (b) (frozen section). Intense Prussian blue reaction product is seen in the coronary wall (d and f) when AlumHemFeI–T is used. The endothelial and medial layers (arrow in f) and endothelial cell nuclei (arrowheads in f) appear intensely stained. For negative controls see Supplementary Fig. S3. Figure 5. Sections of myocardium from mice perfused with PTA (a), I2KI (b), AlumHemFeI (c) and AlumHemFeI-T (d). The images in (e) and (f) are magnifications of the regions outlined in c, and d respectively. The purple colour in (a) indicates the presence of PTA within the myocardium and coronary wall. Intense iodine staining of the myocardium is seen in (b) (frozen section). Intense Prussian blue reaction product is seen in the coronary wall (d and f) when AlumHemFeI–T is used. The endothelial and medial layers (arrow in f) and endothelial cell nuclei (arrowheads in f) appear intensely stained. For negative controls see Supplementary Fig. S3. imaged. Nonetheless, Figs 4b and 5b confirm that the CT enhancement seen in Fig. 2b is due to I2KI accumulation within the entire aortic wall and the myocardium. Figures 4c,d and 5c,d suggest that, interestingly, AlumHemFeI and AlumHemFeI-T stain specific compo- nents of the vessel wall. The Prussian blue reaction product showed the distribution of iron (in the aluminum, iodine, and iron heavy element mixture) as different intensities of blue. For AlumHemFeI, iron was present within the interlamellar units of the aorta (Fig. 4c,e and Supplementary Fig. S2), with the highest concentra- tion in the endothelium (Fig. 4e). Discussion p p y y Further contrast enhancement was achieved when perfusion with AlumHemFeI CT stain was preceded by flushing with a saline solution of the non-ionic detergent Triton™ X-100, resulting in greater permeation of the stain into the vessel wall. We suspect that the saline-Triton™ X-100 solution allowed access to binding sites in the tissues that would not normally be accessible to the stain. The use of Triton™ X-100 also resulted in sharper delineation of the vessel wall in the micro-CT images, likely because of the increased permeability of the vessel wall to the final saline flush, thereby removing any excess stain that is present in surrounding tissue. il , y g y p g Based on the AlumHemFeI-T histology and the micro-CT results (Figs 2, 4, 5 and Table 1), we speculate that the non-ionic detergent is acting in two ways. First, the cell membranes of endothelial tissue are disrupted when exposed to the detergent, thereby allowing more stain to be delivered into the vessel wall. This assumption is sup- ported by the more localized enhancement of contrast in the vessel wall compared to the surrounding tissue in the micro-CT images and the increased intensity of the blue colour observed in the Prussian blue-stained histology sections (compared to those stained with AlumHemFeI). Second, the increased permeability allowed for a more effective flush of the CT stain during the final saline perfusion stage; excess CT stain that wasn’t bound to tissue was removed resulting in more uniform enhancement of the vessel walls and sharper vessel wall delineation in the micro-CT images. The observed CT contrast is lower because of the partial-volume effect (where the CT voxels are larger than the thickness of the vessel wall). g Other groups have reported that using a graded ethanol biochemical conditioning technique enhanced the permeability of murine tissues29 and provided good soft tissue contrast without the use of a CT stain23. However, it has also been shown that when applied to both formalin-fixed and unfixed collagen-containing tissue, ethanol causes large amounts of shrinkage36–38. Our results show that using a biological detergent prior to perfusion of unfixed tissue permeabilizes the cell endothelial membrane and potentially opens channels in other tissue com- ponents to allow for greater stain penetration without the drying effects of ethanol. p g p y gf AlumHemFeI and AlumHemFeI-T contain three highly attenuating elements – iron, iodine, and aluminum. Discussion In this study, we have shown that a novel, highly x-ray attenuating stain, AlumHemFeI-T, delivered via vascular perfusion, allows for whole-body ex vivo visualization of the vessel wall over the entire vascular tree in mice using micro-CT. The perfusion/staining technique promises to provide valuable information to pre-clinical cardiovas- cular studies due to the ease of staining and wide availability of laboratory micro-CT scanners. Optical histology verified that AlumHemFeI-T has an affinity for endothelial cells and the contents within the interlamellar units in the aorta and the media of the coronary arteries. Vessels were also clearly visible in the CT images of the cerebral vasculature and of highly vascularized organs such as the liver and kidney. The AlumHemFeI stain is inert and has no known toxic properties, unlike the osmium tetroxide stain that has previously been used to study the coronary arteries in mice via whole-body perfusion22. y p Although an original consideration for this study was the potential to stain elastin, perfusing the animals with traditional Verhoeff’s elastin stain resulted in intravascular precipitation, limiting adequate perfusion. Some of the Fe3+ in Verhoeff’s staining solution oxidizes heamatoxylin to hematein, and is reduced to Fe2+. The hematein and iron then form complexes that bind to all components of tissues. The iodine in the solution may, by an unknown mechanism, increase the entry of iron-hematein complex into elastin, which is a hydrophobic protein. After immersion in Verhoeff’s solution, tissue sections are partly destained in aqueous ferric chloride, which extracts iron-hematein from cytoplasm and collagen, leaving the black complex in cell nuclei, elastic fibers and laminae, and myelinated nerve fibers32. When Verhoeff’s solution was perfused through mice, precipitates, prob- ably from interaction of proteins and inorganic salts with the ethanol solvent, blocked the microvasculature. This rendered traditional Verhoeff’s incompatible with whole body perfusion and necessitated modification of the stain. We successfully avoided vessel blockage by replacing the alcoholic heamatoxylin used in the traditional Verhoeff’s stain with aluminum (Harris) heamatoxylin (AlumHemFeI). Although the AlumHemFeI stain does not bind to elastin, it may be used to provide a surrogate stain of elastin by binding to components in the interla- mellar units. The AlumHemFeI stain achieved more micro-CT contrast enhancement of all vessel walls than I2KI perfusion, especially those of the coronary arteries and aorta. Results Q lit t Particularly noteworthy was the finding of intense blue also seen within some endothelial-cell nuclei (Fig. 4e and Supplementary Fig. S2). Within the heart (Fig. 5c,e and Supplementary imaged. Nonetheless, Figs 4b and 5b confirm that the CT enhancement seen in Fig. 2b is due to I2KI accumulation within the entire aortic wall and the myocardium. Figures 4c,d and 5c,d suggest that, interestingly, AlumHemFeI and AlumHemFeI-T stain specific compo- nents of the vessel wall. The Prussian blue reaction product showed the distribution of iron (in the aluminum, iodine, and iron heavy element mixture) as different intensities of blue. For AlumHemFeI, iron was present within the interlamellar units of the aorta (Fig. 4c,e and Supplementary Fig. S2), with the highest concentra- tion in the endothelium (Fig. 4e). Particularly noteworthy was the finding of intense blue also seen within some endothelial-cell nuclei (Fig. 4e and Supplementary Fig. S2). Within the heart (Fig. 5c,e and Supplementary Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 6 www.nature.com/scientificreports/ Fig. S3), very little iron signal was observed in the myocardium and the wall of the coronary artery, but again with the highest concentration in the endothelial lining of the coronary.h Fig. S3), very little iron signal was observed in the myocardium and the wall of the coronary artery, but again with the highest concentration in the endothelial lining of the coronary.h g g y The sections from AlumHemFeI-T-perfused mice indicate that a substantial amount of iron was present across the entire aortic wall (Fig. 4d and Supplementary Fig. S2), with particularly striking signal in the endothe- lial cell nuclei (Fig. 4f). Iron was also present in the interlamellar units, especially the innermost unit, as well as the adventitia (Fig. 4d,f). Figure 5d shows the myocardium and representative coronary artery, where the blue reaction product is present in the endothelial and medial layers of the coronary wall, again with remarkably intense staining of the endothelial cell nuclei (Fig. 5d,f).hi g g These staining profiles for the AlumHemFeI- and AlumHemFeI-T-perfused mice, in both aorta and coronary artery, suggest a notable enhancement of stain delivery, including into the endothelial cell nucleus itself, when the detergent Triton™ X-100 is employed in the stain delivery protocol. Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z Discussion The histological study shows that iron is present in select tissues and we have used this information to confirm the presence of the CT stain in those tissues. However, further analysis is needed to determine which of the three attenuating elements are dominantly contributing to the micro-CT contrast. Preliminary studies where mice were perfused with combinations of the three stain ingredients (FeCl3, I2KI, and Harris heamatoxylin) demonstrated CT enhancement only when iodine was present in the perfusate (results not shown), suggesting that most of the CT attenuation may be caused by iodine. In future studies, the actual amount of each elemental component in the various tissues will be determined using inductively coupled plasma mass spectrometry (ICP-MS), which is capable of detecting the presence of aluminum, iron and iodine in tissue. Scientific REPOrts \| (2019) 9:698 \| DOI:10.1038/s41598-018-36905-z 7 www.nature.com/scientificreports/ Soft tissue staining with various CT stains can provide morphological information and has been explored extensively by soaking of tissues and organs. Our group pioneered the use of vascular perfusion to target the delivery of the CT stain to vascular tissue – by taking advantage of the inherent permeability of fresh, unfixed tissue to the small molecules in the CT stains – while simultaneously speeding up preparation from days to min- utes24. In the earlier study, we demonstrated that PTA stains the vasculature, while I2KI is useful for the study of cardiac tissue. We now provide clearer understanding of the localization of each of these stains. PTA enhanced the contrast of the myocardium, aorta and the parenchyma of the liver in the micro-CT images (Fig. 1). This observation is consistent with previous findings that have demonstrated the affinity of PTA for collagen and the extracellular matrix34,25, and is further validated by the histological analysis (Figs 4 and 5). Although the pres- ence of PTA in the vessel walls and heart is evident, the CT enhancement is lower than that seen with other CT stains evaluated (I2KI, AlumHemFeI, and AlumHemFeI-T). I2KI provides greater CT contrast enhancement of the myocardium and ascending aorta, allows visualization of the myocardial fibres and provides a negative lumen stain of the coronary arteries in micro-CT images. y g In this study, we also introduce the use of a controlled CT-stain perfusion system, which enables physiological pressurization and precise timing of all the steps of stain delivery. Conclusions We have developed and demonstrated a novel non-toxic stain – AlumHemFeI-T – that provides excellent visual- ization of the vasculature of mice for virtual histology, with unique sensitivity to endothelial nuclei as well as the media of large and medium size vessels. Delivered using a novel perfusion apparatus, the AlumHemFeI-T stain represents a promising new tool for scientists studying both normal and diseased vasculature in rodent models. Discussion While similar results can likely be achieved using hand-perfusion or a gravity-fed IV set up, the use of timed perfusion and known pressure resulted in higher reproducibility of the results. Furthermore, in this study, animals were perfused using a retrograde abdominal aortic procedure with cannulation occurring just below the kidneys. This route was selected to enable the visu- alization of the coronary as well as the thoracic vasculature, although it does not allow for the lower abdominal region to be perfused. Alternative stain delivery routes, such as left ventricular puncture or antegrade abdominal aortic perfusion would allow for other vascular systems to be perfused and studied. Many disease models that involve the changes or alterations in the blood vessel wall (e.g. aneurysm models, atherosclerosis models, cancer, angiogenesis, and vascular grafts) would benefit from this technique2,5,7. g g gti q Larger animals are also commonly used in studies of vascular disease and we expect that the perfusion with AlumHemFeI-T, as described in this work, will be equally applicable to such studies. The perfusion technique uses the vasculature as a conduit to deliver the stain to the vessel walls and tissues, which can have varying properties across different species. Hence, some optimization to the perfusion times may be required in different animal species, body weights, or tissue type. Nevertheless, preliminary perfusion of a guinea pig, an adult rat and an explanted porcine heart demonstrated similar results as those observed in mice. (Supplementary Fig. S4 shows an image of the perfused rat). While not demonstrated in this study, the presented virtual histology technique also allows for high-resolution non-destructive imaging of tissues excised from the perfused animals (i.e. sectioned post fixation). However, further investigation is required to determine if stain diffusion over long fixation and scan periods will affect stain localization. pf Our rapid perfusion and staining technique allows for the heavy element stains to be quickly and evenly dis- tributed throughout the tissues addressing limitations that other groups have faced and allowing for larger sample sizes to be stained and studied. Moreover, tissues stained with AlumHemFeI-T can then be harvested from the intact mouse and further processed histologically, an important advantage over other CT staining strategies that allows for multi-modality assessment of the vasculature. References 1. Elkerton, J. S., Xu, Y., Pickering, J. G. & Ward, A. D. 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Acknowledgementsh This work was funded by a grant from the Heart and Stroke Foundation of Canada (G-14-0005959). M.D. is a Career Investigator of the Heart and Stroke Foundation of Ontario. Funding support also came from the Canadian Institutes of Health Research (FDN-143326). J.G.P. holds the Heart and Stroke Foundation of Ontario Barnett-Ivey Chair at the Robarts Research Institute. C. Cruje acknowledges funding from an Ontario Graduate Scholarship. JAK thanks the Biological Stain Commission for a grant in aid of research. The authors also thank C. Crukley for helpful discussion and assistance with early histological studies. Author Contributions P.J.D.-B., Z.N., J.G.P., J.A.K. and M.D. conceived the experiments. P.J.D.-B., Z.N. and C.C. conducted the experiments; P.J.D.-B. and C.C. wrote the manuscript. Z.N. prepared and stained the histological sections. J.A.K. and J.J.L. stained some of the histological sections for analysis. C.C., Z.N., J.G.P and M.D. prepared the figures. 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https://openalex.org/W2346474363	https://europepmc.org/articles/pmc4858763?pdf=render	English	null	Touch uses frictional cues to discriminate flat materials	Scientific reports	2,016	cc-by	6,393	David Gueorguiev1, Séréna Bochereau2, André Mouraux1, Vincent Hayward2 & Jean-Louis Thonnard1,3 received: 14 January 2016 accepted: 18 April 2016 Published: 06 May 2016 In a forced-choice task, we asked human participants to discriminate by touch alone glass plates from transparent polymethyl methacrylate (PMMA) plastic plates. While the surfaces were flat and did not exhibit geometric features beyond a few tens of nanometres, the materials differed by their molecular structures. They produced similar coefficients of friction and thermal effects were controlled. Most participants performed well above chance and participants with dry fingers discriminated the materials especially well. Current models of tactile surface perception appeal to surface topography and cannot explain our results. A correlation analysis between detailed measurements of the interfacial forces and discrimination performance suggested that the perceptual task depended on the transitory contact phase leading to full slip. This result demonstrates that differences in interfacial mechanics between the finger and a material can be sensed by touch and that the evanescent mechanics that take place before the onset of steady slip have perceptual value. Tactile sensing plays a key role in our interaction with the world around us. We constantly need to assess the nature of the objects we are in contact with in order to go about our daily lives. Thus far, it has been shown that many manipulative and perceptual tasks are performed on the basis of gross shape1,2 of frictional properties3 and of surface topography4,5. Tactile mechanics provide cues about the physical properties of touched objects, includ- ing their shape6, their softness7 or their friction8. Therefore, objects made of different materials but having similar shapes, frictional properties, and surface topography represent a challenge for the sense of touch. Under these conditions, objects are generally assumed to be discriminated on the basis of their mass properties or thermal properties9,10. To our knowledge, however, it was never suggested that differences in the materials we touch could be detected on the basis of molecular-level properties. Water is almost universally present in our environment. With rare exceptions, all surrounding objects are naturally coated with water and water represents approximately 60% of the mass of our bodies. Water has specific molecular-level bonding properties, which manifest themselves by the hydrophobic or hydrophilic character of materials. For example, the dew on grass leaves owes its existence to the hydrophobicity of the leaves. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Institute of Neuroscience, Université catholique de Louvain, B-1200, Brussels, Belgium. 2Sorbonne Universités, UPMC Univ Paris 06, UMR 7222, ISIR, F-75005, Paris, France. 3Cliniques Universitaires Saint-Luc, Physical and Rehabilitation Medicine Department, Université catholique de Louvain, B-1200, Brussels, Belgium. Correspondence and requests for materials should be addressed to J.-L.T. (email: jean-louis.thonnard@uclouvain.be) Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 Touch uses frictional cues to discriminate flat materials David Gueorguiev1, Séréna Bochereau2, André Mouraux1, Vincent Hayward2 & Jean-Louis Thonnard1,3 David Gueorguiev1, Séréna Bochereau2, André Mouraux1, Vincent Hayward2 & Jean-Louis Thonnard1,3 David Gueorguiev1, Séréna Bochereau2, André Mouraux1, Vincent Hayward2 & Jean-Louis Thonnard1,3 The skin’s stratum corneum, which covers our fingers, is mainly hydrophobic but also exhibits permeation properties11 and moisture endows it with peculiar frictional properties that influence its contact mechanics with surfaces12.i l Most previously studied surface-related tactile perceptual tasks rely on sliding fingers on surfaces because discrimination performance is dramatically improved during dynamic exploration13. The primary reason for this enhanced performance is that the mechanical fluctuations induced by sliding bring rich information about the underlying topography of a surface compared to the minute skin deformations induced by static contacts. As shown later, in the limit case of perfectly flat surfaces, complex mechanical fluctuations still take place for a large range of loads and sliding velocities. These fluctuations take place during sliding as well as during the transitory phases between sticking and slipping.i Recent studies have shown that the transition from static to dynamic finger-glass contacts can lead to surpris- ingly complicated dynamics where contacts can fail progressively or catastrophically under load14–16 owing to the nonlinear nature of friction and skin biomechanics. Here, we hypothesized that this dynamics could enable humans to discriminate between surfaces that are identical in all their physical aspects, except their molecular structure.l We tested this hypothesis in two experiments where we asked human participants to discriminate between flat surfaces made of glass or made of polymethyl methacrylate (PMMA) (see Supplementary Fig. 1). The samples 1Institute of Neuroscience, Université catholique de Louvain, B-1200, Brussels, Belgium. 2Sorbonne Universités, UPMC Univ Paris 06, UMR 7222, ISIR, F-75005, Paris, France. 3Cliniques Universitaires Saint-Luc, Physical and Rehabilitation Medicine Department, Université catholique de Louvain, B-1200, Brussels, Belgium. Correspondence and requests for materials should be addressed to J.-L.T. (email: jean-louis.thonnard@uclouvain.be) Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 1 1 www.nature.com/scientificreports/ Figure 1. Psychophysical measurements. (a) Performance at the task presented as Mean ± s.d. for experiment 1. (b) Performance at the task presented as Mean ± s.d. in experiment 2. (c) The mean difference of temperature between the fingertip and the samples plotted against performance for all participants of both experiments. (d) Mean moisture level of the fingertip plotted against individual performance for all participants of both experiments. Figure 1. Psychophysical measurements. (a) Performance at the task presented as Mean ± s.d. for experiment 1. (b) Performance at the task presented as Mean ± s.d. in experiment 2. David Gueorguiev1, Séréna Bochereau2, André Mouraux1, Vincent Hayward2 & Jean-Louis Thonnard1,3 (c) The mean difference of temperature between the fingertip and the samples plotted against performance for all participants of both experiments. (d) Mean moisture level of the fingertip plotted against individual performance for all participants of both experiments. were thermally equalised through bonding to aluminium blocks acting as heat sinks. We established in exper- iment 1 that discrimination between the two materials was possible. In experiment 2, we reproduced the task and recorded the contact forces with exacting accuracy together with the psychophysical performance of the participants. The two experiments differed also in the thermal conditions. In experiment 1, the temperature of the samples was kept constant at a value near the natural fingertip temperature. In experiment 2, the samples were at room temperature. Results (a) The rate of change of the tangential force component enabled the extraction of the objective markers t1, t2, t3 and t4 (see Materials and Methods), which were used to define the different phases within a swipe: loading phase (t0-t1), partial slip (t1-t2) and full slip (t2-t3). (b) The coefficient of dynamic friction, Mean ± s.d., was not distinguishable between materials. The 10 pairs of symbols represent the glass (red) and PMMA (blue) values for each participant. Although the overall individual performance for both materials is identical for the two datasets, the data for glass are slightly shifted downwards for better visibility. dynamic friction. (a) The rate of change of the tangenti Figure 2. Trial segmentation and coefficient of dynamic friction. (a) The rate of change of the tangential force component enabled the extraction of the objective markers t1, t2, t3 and t4 (see Materials and Methods), which were used to define the different phases within a swipe: loading phase (t0-t1), partial slip (t1-t2) and full slip (t2-t3). (b) The coefficient of dynamic friction, Mean ± s.d., was not distinguishable between materials. The 10 pairs of symbols represent the glass (red) and PMMA (blue) values for each participant. Although the overall individual performance for both materials is identical for the two datasets, the data for glass are slightly shifted downwards for better visibility. starting point of each exploration to ensure that the samples were explored from the most distal to the most proximal. The participants’ movements were constrained in order to allow the collection of mechanical data. Each individual sample was explored from left to right in a single swipe. Participants could repeat the exploration up to five times per trial before reporting a judgment. Despite these constraints, performance was also highly significant (one sample t-test: t(11) = 4.16, p = 0.0016, 95% CI [6.836, 22.17]). (Fig. 1b) and there was no signifi- cant drop of performance difference between Experiment 1 and 2 (unpaired t-test: t(22) = 1.549, p = 0.136, 95% CI [− 19.92, 2.883 ]). The possibility that detection was mediated by an imperfection of plate surface was also assessed (Supplementary Fig. 3) but the answers showed no bias towards a specific plate.i pp y g pi p We specifically tested for the possibility that discrimination relied on thermal cues. Results To this end, in the two experiments and for each participant we recorded the mean temperature difference between the fingertip and the sample. If there was a substantial possibility for thermal cueing, discrimination performance could be expected to be dependent on the amount of thermal difference between the fingertip and the plates. The Pearson’s corre- lation coefficient between the temperature differences and the individual performances showed non-significant trends corresponding to a slight anti-correlation in experiment 1 (n = 12, R = − 0.30, p = 0.33) in which the samples were warmer and a slight correlation in experiment 2 (n = 10, R = 0.62, p = 0.06) in which the sam- ples were colder. Since the distribution of temperature differences was similar in the two experiments (unpaired t-test: t(20) = 1.386, p = 0.18, 95% CI[− 0.6722, 3.336]), we computed the overall Pearson’s correlation coefficient between the temperature differences and the individual performances, and no correlation was found (n = 22, R = − 0.09 and p = 0.70) (Fig. 1c). These opposite trends for the two experiments rather suggested an effect of the absolute temperature of the fingertip and indeed, higher finger temperature significantly correlated with a better capacity to discriminate the materials (Pearson’ correlation for n = 22, R = 0.47 and p = 0.026). This result was in accordance with previous studies indicating lower mechanical detection thresholds when the fingers are warm17,18. These results are therefore consistent with a discrimination strategy based on mechanical cues, rather than with a strategy based on thermal cues. gy The moisture level of the fingertips was also monitored. We found the moisture level to be strongly anti-correlated with individual performance in experiment 1 (Pearson’s correlation for n = 12, R = − 0.90, p < 0.0001) as well as in experiment 2 (Pearson’s correlation for n = 12, R = − 0.63, p = 0.028). Since the distri- butions were similar (unpaired t-test: t(22) = 0.1875, p = 0.85, 95% CI[− 12.75, 15.29]), we tested the correlation between moisture level and performance across the two experiments. The correlation was robust (Pearson’s cor- relation for n = 24, R = − 0.79, p < 0.0001) (Fig. 1d). Results Experiment 1. Three samples were placed in a single row in front of the participants. Of the three samples, one was always different from the other two. Participants could explore the samples freely as many times as they wanted before reporting which was different from the other two. The samples were kept at a constant temperature similar to the average temperature of the skin (33.4 ± 0.9 °C; mean ± s.d.). Performance at the task was signif- icantly better than chance level (one sample t-test: t(11) = 5.41, p = 0.0002, 95% CI [13.66, 32.38 ]). (Fig. 1a). The results also suggested that participants were better at identifying the PMMA plate among two glass plates as compared to identifying the glass plate among two PMMA plates. We could rule out this bias to be the result of unseen imperfections in their surface because the answers were consistent across the plates and showed no bias towards a specific plate (Supplementary Fig. 2). Experiment 2. The samples were placed in one single column in front of the participant. They were kep room temperature (23.5 ± 1.5 °C; mean ± s.d.). The participant’s hand was guided by the experimenter to Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 2 www.nature.com/scientificreports/ Figure 2. Trial segmentation and coefficient of dynamic friction. (a) The rate of change of the tangential force component enabled the extraction of the objective markers t1, t2, t3 and t4 (see Materials and Methods), which were used to define the different phases within a swipe: loading phase (t0-t1), partial slip (t1-t2) and full slip (t2-t3). (b) The coefficient of dynamic friction, Mean ± s.d., was not distinguishable between materials. The 10 pairs of symbols represent the glass (red) and PMMA (blue) values for each participant. Although the overall individual performance for both materials is identical for the two datasets, the data for glass are slightly shifted downwards for better visibility. Figure 2. Trial segmentation and coefficient of dynamic friction. (a) The rate of change of the tangential f bl d h i f h bj i k d ( M i l d M h d ) Figure 2. Trial segmentation and coefficient of dynamic friction. Results The strong influence of fingertip moisture on performance discrimination motivated us to investigate further the connection between the perceptual performance and the underlying contact mechanics.ii We first verified that, during non-constrained exploration, the behavioural parameters (speed of exploration, normal force applied) did not correlate with individual performance (see Supplementary Fig. 4). We then seg- mented each swipe into three epochs: a loading phase during which the finger contact is stuck, a partial slip phase during which the outer part of the contact area starts slipping14 and a full slip phase during which the contact area is fully sliding (Fig. 2a and materials and methods). The aim of this segmentation was to determine which epoch provided the greatest differentiation between the materials being touched.fii f We computed the dynamic coefficient of friction for the two materials defined as the ratio of the magni- tude of tangential and normal forces generated by the interaction during full slip for all trials (Fig. 2b). The values of the coefficient of friction varied across participants with a mean value of 1.13 ± 0.13 (mean ± s.d.) but a Mann-Whitney test with a Bonferroni correction did not show significant intra-individual differences related to Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 3 www.nature.com/scientificreports/ Figure 3. Analysis of the frictional fluctuations. (a) Comparison of the amplitude spectra of the tangential force component in the spatial domain. Linear regression gave parameters α and logβ . The differences Δ α and Δ logβ between the mean spectra of the two materials were computed for all participants. (b–d) The differences in the frictional noise \|Δ logβ \| between glass and PMMA plotted for each phase against performance: (b) Partial slip. (c) Loading phase. (d) Full slip. Figure 3. Analysis of the frictional fluctuations. (a) Comparison of the amplitude spectra of the tangential force component in the spatial domain. Linear regression gave parameters α and logβ . The differences Δ α and Δ logβ between the mean spectra of the two materials were computed for all participants. (b–d) The differences in the frictional noise \|Δ logβ \| between glass and PMMA plotted for each phase against performance: (b) Partial slip. (c) Loading phase. (d) Full slip. the materials: nine participants had p-values > 0.05 and one participant, whose performance was average (42,9% of correct answers) had a p-value = 0.042. Discussion Performance within our sample of participants was significantly above the chance level in the two experiments, showing that tactile discrimination between two flat materials is possible even in the absence of thermal and surface topography differences. If flat surfaces could be distinguished on the sole basis of frictional cues, then dif- ferences must have arisen from molecular-scale properties influencing the interfacial contact of the skin-material pair, in turn influencing its frictional dynamics. The moisture content of the fingers strongly correlated with performance across the study, suggesting that the interaction of water either with the skin tissue or with the skin-material interface played an important role in the tactile discrimination of these two flat surfaces.ffi p y pl During steady sliding, there was no measurable difference in contact mechanics (coefficient of friction, spec- tral properties of force fluctuations) between the two materials. In contrast, there were significant differences in the magnitude of the interfacial force variations during the partial slip phase corresponding to a progressively failing contact. The partial slip phase comprises a mix of adhesion and slip where, typically, an annulus of failure grows from the periphery of the contact region to eventually invade the whole contact14 (see also Supplementary Movie 1). The present measurements corroborated the earlier observation that partial slip tends to last from 100 ms to 300 ms in finger-glass contacts15. During this period, the contact evolution can sometimes take the form of a catastrophic failure once a critical load is reached. This finding suggests that the evolution of the contact state, owing to the nonlinearity of friction, can be critically sensitive to small changes in the frictional behaviour of the skin-surface interface.hfh The observed macroscopic effects must take their roots in smaller-scale physics. The moisture level of the fingertip was already shown to dramatically affect the time course of contact failure under load and thus could explain the performance dependency that we observed between fingertip moisture and discrimination perfor- mance14. The surfaces that we employed in the present study differed by their hydrophobicity. Ordinary uncoated glass has a hydrophilic, amorphous solid atomic structure and PMMA is a hydrophobic transparent polymer. The other material of the contact pair, the skin stratum corneum, is mostly made of keratin, a natural glassy polymer with strong affinity with water. As reviewed recently22,23, water plasticizes the stratum corneum and reduces its elasticity leading to combined adhesion mechanisms. Discussion A first mechanism is associated with capillary forces at small normal loads and short time scales24, a second is due to plasticization for longer time scales25, and others to molecular interactions. The difference in material hydrophobicity is likely to affect these phenomena differen- tially and their relative contributions could impact the evolution of the stick-to-slip dynamics. At onset, the finger would tend to stick more on glass than on plastic owing to a stronger influence of capillary forces in the early stages of a tangentially loaded contact. Subsequently, during the partial slip phase, higher magnitude of frictional dynamics for PMMA could arise from the rate of breaking of the molecular bonds during the progressive failure of adhesion, which is akin to crack formation. Plasticization occurs over a longer time course and it is unlikely to play a direct role during the early transient phase of tactile exploration but the free exploration allowed in exper- iment 1 also took place on a longer time course than single swipes. Therefore, different plasticization dynamics might be responsible for the difference in discrimination performance between the two materials, which we observed in experiment 1. The strong effect of hydration of the fingertip could also influence the transmission of subtle frictional fluctuations through variations in skin damping.h l g p g The onset of slip received no attention until now since it was hitherto assumed that the tactile perception of materials relied predominantly on interactions taking place over extended periods of full slip. Classical studies of touch focus almost exclusively on rigid surface features without consideration of chemistry of the materials26. Our results suggest that other properties affecting frictional dynamics must be taken into consideration, and jus- tifies the current shift toward studies involving naturalistic surfaces27,28. Moreover, recent reports of astonishing tactile discrimination performance with textured surfaces at the nanometre scale29 could be explained, not by direct differences in topography among these surfaces, but rather by the impact that small scale features have on interfacial frictional dynamics. Results The coefficients of friction between the two materials were significantly anti-correlated with individual performance (Pearson’s correlation for n = 10, R = − 0.67, p = 0.036): discrimi- nation performance was, on average, better when the difference in coefficient of friction was small. This indicates that discrimination did not rely on differences in the average dynamic coefficient of friction during full slip, but does suggest a possible role of frictional dynamics during other phases of the exploration. We thus analysed the spectral characteristics of the forces during the different phases of the sliding. For this purpose, the signals were analysed in the spatial domain such as to characterize the fine fluctuations of force independently of the net slip velocity19,20. As typically is the case, the spatial frequency amplitude spectrum followed a power law relationship21, T = β η α, where β is a magnitude factor and α quantifies the decay of amplitude with frequency (Fig. 3a).h g qi y p q y g The spectra were averaged within participants for all trials and the magnitudes and decays were estimated. For each of the three epochs, the spectrum in the range 10−1− 10 mm−1 was compared between glass and PMMA. There was no significant difference in spectral decay between the materials but the magnitudes differed (Δ logβ , Fig. 3b–d). Discrimination performance was strongly correlated with \|Δ logβ \| during partial slip (Pearson’s corre- lation for n = 10, R = 0.83, p = 0.003) and moderately during the loading phase (Pearson’s correlation for n = 10, R = 0.70, p = 0.023). On the other hand, correlation was very weak during fully developed slip (Pearson’s cor- relation for n = 10, R = 0.007, p = 0.99). These correlations between the transient frictional dynamics observed during the early stages of a swipe and individual performance suggest that, during these stages, frictional dynam- ics of the interactions between the fingertip and the two materials differed, and that this difference enabled the participants to discriminate the two materials. Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 4 www.nature.com/scientificreports/ Materials and Methods The force plate meas- ured the normal force components using two load cells (9313AA1, Kistler AG, Switzerland) and the tangential force component using one load cell (9217A, Kistler AG, Switzerland). The offset of the force plate was 1.0 mN for the tangential component of the force and 40 mN for the normal component. The signals were digitized at 10 kHz. The location of the fingertip contact on the plate was estimated by computing the position of the centre of pressure. The force transducer temporarily lost calibration during the study. As a result, the force data of two participants could not be analysed. Analysis of the contact mechanics. Detailed force analyses were performed on swipes for which the continuous motion on the surface was longer than one second and the exploration length was at least four centi- metres. The continuous force measurements were segmented into single epochs corresponding to distinct phases within single swipes (Fig. 2a). To express the variations in tangential force during each swipe, the tangential force component was smoothed using a moving average filter with a 0.25 s time constant, and then differentiated. The onset of exploration (t0) was defined as the instant when the normal force reached a value greater than the max- imum noise level of the force plate (40 mN). This time point corresponds to the moment when the force sensor measures a significant increase of normal force due to the finger pressing against the surface. The end of the exploration (t4) was defined as the instant when the first derivative of the tangential force reaches its minimum. This corresponds to the moment when the participant begins unloading the finger from the surface. Three suc- cessive segments were then defined (loading phase, partial slip and full slip). The loading phase segment started at the onset of exploration (t0). During this phase, the fingerpad remained in static contact with the surface, and an accelerating increase in tangential force was observed. The transition between the loading phase segment and the partial slip segment was defined as the instant when the rate of the tangential force reached a maximum (t1), as this sudden change in tangential force dynamics was thought to result from the occurrence of partial slip between the fingerpad and surface. Materials and Methods The thermocouples were located under the plates (OS36, OMEGA engineering Inc.) and controlled by a thermo-controller (MICROMEGA CN77000, OMEGA engineering Inc.) in order to keep the temperature of the samples constant throughout the whole experiment and match the average temperature of the human fingertip. This procedure could not be implemented in experiment 2, because the setup for temperature control could not be combined with the force sensing plates used for the fine measurements of force dynamics. General procedure. The procedure was a forced choice task with three alternatives (3-AFC) where one sample differed from the other two (“odd-man-out”). This procedure was chosen because we did not have prior assumption of the relevant tactile information that could be used to discriminate the surfaces, and it encouraged the participants to search for any subtle differences. The participants washed their hands with commercial liquid soap and waited five minutes before starting the task in order to stabilize the frictional properties of the skin. Three randomly placed samples were presented to the participant (either two samples of PMMA and one sample of glass, or one sample of PMMA and two samples of glass). The participants explored the samples with the index finger of their dominant hand and reported which of the three samples differed from the others two. At the begin- ning of the experiment, the samples were cleaned with commercial cleaner for glasses (Si Clair, SI International, France) and checked for imperfections. They were additionally quickly cleaned with microfiber cloth every three trials. The performance of each participant was given by the percentage of correct answers and participants were asked about possible imperfections on the samples. An experiment comprised three blocks of fourteen trials. Before and after each block, the temperature of the samples and of the fingertip was assessed with an infrared thermometer (Raytek MI3-M, Raytek, USA). In experiment 2, the temperature values could not be collected for two participants because of a temporary unavailability of the recording device. The moisture level of the fingertip was also measured by averaging three repeated measures using a corneometer (CM 825, Courage + Khazaka electronic GmbH, Germany). Force measurements. In experiment 2, the contact forces between the finger and the samples were recorded by a sensitive force sensing plate designed in-house on which the samples were clamped. Materials and Methods The ethics committee on human research of Université catholique de Louvain approved the study (Virtual Prototyping of Tactile Displays, PROTOTOUCH-317100). All participants gave written informed consent. The investigation conformed to the principles of the Declaration of Helsinki and experiments were performed in accordance with relevant guidelines and regulations. Participants. Data were collected from 24 healthy volunteers (12 in experiment 1 and 12 in experiment 2) aged between 22 and 61. Participants were blindfolded and white noise was played at a comfortable listening level through headsets in order to mask auditory cues. Materials. The samples were made of ordinary glass (clear glass, Leroy Merlin, France) or of polymethyl methacrylate (PMMA) plates (Plexiglas®, ABP Beaumont plastique, France). Surfaces were perfectly flat. They were chosen for their differences in atomic structure and hydrophobicity. The topographical profiles were assessed with a digital holographic microscope (Lyncée Tec, Switzerland) to check the flatness and absence of topographic features (see Supplementary Fig. 1). The samples dimensions were 60 × 30 × 2 mm. According to findings from previous studies, the differences in the thermal properties of PMMA (heat capacity: 1460 j/kg, thermal conductivity: 0.2 j/m, Contact Coefficient: 0.6 103 J/m2s1/2K) and glass (heat capacity: 837 j/kg, thermal conductivity: 0.78 j/m, Contact Coefficient: 1.3 103 J/m2s1/2K) were insufficient to provide thermal cues for the discrimination of the samples or noticeable thermal changes to the fingertip9,30,31. Furthermore, the temperatures of the samples were kept equalised by bonding all triplets of samples to 10 mm thick aluminium blocks acting as heat sinks. The edges were protected with adhesive tape to prevent the participants from using them as a source of information. In experiment 1, the samples were warmed using an infrared heat lamp and thermocouples. Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 5 www.nature.com/scientificreports/ The thermocouples were located under the plates (OS36, OMEGA engineering Inc.) and controlled by a thermo-controller (MICROMEGA CN77000, OMEGA engineering Inc.) in order to keep the temperature of the samples constant throughout the whole experiment and match the average temperature of the human fingertip. This procedure could not be implemented in experiment 2, because the setup for temperature control could not be combined with the force sensing plates used for the fine measurements of force dynamics. Materials and Methods The transition from partial slip to full slip was defined as the instant (t2) when the derivative of the tangential force became smaller than its standard deviation (estimated in the interval [t1, t4]). This was justified by the fact that once the finger starts sliding steadily against the surface, the tangential force remains relatively constant, provided that both the normal force and the dynamic coefficient of friction stay constant. Finally, the end of the full slip segment was defined as the time point (t3) when the derivative of the tangential force became more negative than its standard deviation estimated in the interval [t1, t4]. This last time point marked the end of the phase during which the finger was steadily sliding against the surface. Since the speed of exploration varied between trials, the force data measurements were converted to the spatial domain20. After this transformation, the length of the full slip segments were further restricted to two centimetres of sliding in order to match their duration with that of the transient phases. Since averaging the Spatial Fast Fourier Transform (SFFT) over all swipes for each participant required using identical epoch for each swipe, we computed the t0, t1 and t2 values for each participant over all its swipes, turned them into spatial coordinates and used their median values to implement the participant’s average SFFT of glass and PMMA. Statistics. The decision to use parametric or non-parametric statistical methods on given data sample was motivated by the D’Agostino and Pearson omnibus normality test, which we performed on all analysed samples using GraphPad Prism software. J , ( ) 2. Kappers, A. M. L. Human perception of shape from touch. Philos. Trans. R. Soc. B Biol. Sci. 366, 3106–3114 (2011). References 1. Jenmalm, P. & Johansson, R. S. Visual and Somatosensory Information About Object Shape Control Manipulative Fingertip Forces. J. Neurosci. 17, 4486–4499 (1997). Author Contributions D.G., S.B., A.M., V.H. and J.-L.T. designed the study, D.G. and S.B. performed the research, D.G. and J.-L.T nalysed the data, D.G., S.B., A.M., V.H. and J.-L.T. wrote the paper. D.G., S.B., A.M., V.H. and J.-L.T. designed the study, D.G. and S.B. performed the research, D.G. and J.-L.T. analysed the data, D.G., S.B., A.M., V.H. and J.-L.T. wrote the paper. References Some Parameters Affecting Tactile Frictio i 4. Dinç, O. S., Ettles, C. M., Calabrese, S. J. & Scarton, H. A. Some Parameters Affecting Tactile Friction. J. Tribol. 113, 512 (1991). 5 Ad M J B i B J & J h S A F i i d l b i i f h ki T ib l L tt 26 239 253 (2007) ̧f 25. Adams, M. J., Briscoe, B. J. & Johnson, S. A. Friction and lubrication of human skin. Tribol. Lett. 26, 239–253 (2007).h 6. Jones, L. A. & Lederman, S. J. Human Hand Function. The Journal of Hand Surgery European Volume 32, 280, doi: 10.1016/j jhse.2007.04.015 (2006). j 7. Bergmann Tiest, W. M. & Kappers, A. M. L. Analysis of haptic perception of materials by multidimensional scaling and physica measurements of roughness and compressibility. Acta Psychol. (Amst). 121, 1–20 (2006). 8. Weber, A. I. et al. Spatial and temporal codes mediate the tactile perception of natural textures. Proc. Natl. Acad. Sci. USA 110 17107–12 (2013). ( ) 29. Skedung, L. et al. Feeling small: exploring the tactile perception limits. Sci. Rep. 3, 2617 (2013). 0. Jones, L. A. & Berris, M. Material discrimination and thermal perception. 11th Symp. Haptic Interfaces Virtual Environ. Teleoperato Syst. 2003. HAPTICS 2003. Proceedings. (2003). doi:10.1109/HAPTIC.2003.1191267. y g 1. Ho, H. & Jones, L. A. Material identification using real and simulated thermal cues. Conf. Proc. IEEE Eng. Med. Biol. Soc. 4 2462–2465 (2004). References References 1. Jenmalm, P. & Johansson, R. S. Visual and Somatosensory Information About Object Shape Control Manipulative Fingertip Forces. J. Neurosci. 17, 4486–4499 (1997). 2. Kappers, A. M. L. Human perception of shape from touch. Philos. Trans. R. Soc. B Biol. Sci. 366, 3106–3114 (2011). J ( ) 2. Kappers, A. M. L. Human perception of shape from touch. Philos. Trans. R. Soc. B Biol. Sci. 366, 3106–3114 (2011). Scientific Reports \| 6:25553 \| DOI: 10.1038/srep25553 6 www.nature.com/scientificreports/ 3. Westling, G. & Johansson, R. S. Factors influencing the force control during precision grip. Exp. brain Res. 53, 277–84 (1984).h gl g g p g p p 4. Bensmaïa, S. & M. H. The Vibration of texture. Somatosens. Mot. Res. 20, 33–43 (2003). h 5. Manfredi, L. R. et al. Natural scenes in tactile texture. J. Neurophysiol. 111, 1792–802 (2014). 6. Goodwin, a W., John, K. T. & Marceglia, A. H. Tactile discrimination of curvature by humans using only cutaneous information from the fingerpads. Exp. brain Res. 86, 663–72 (1991).t i g p p , ( ) 7. Srinivasan, M. A. & Lamotte, R. H. Tactual discrimination of softness. J. Neurophysiol. 73, 88–101 (1995). i g p p ( ) 7. Srinivasan, M. A. & Lamotte, R. H. Tactual discrimination of softness. J. Neurophysiol. 73, 88–101 (1995). j g p y 9. Ho, H.-N. & Jones, L. A. Contribution of thermal cues to material discrimination and localization. Percept. Psychophys. 68, 118 (2006).f ( ) 0. Tiest, W. M. B. & Kappers, A. M. L. Tactile perception of thermal diffusivity. Atten. Percept. Psychophys. 71, 481–489 (2009).i 10. Tiest, W. M. B. & Kappers, A. M. L. Tactile perception of therm k l f 1. Moser, K., Kriwet, K., Naik, A., Kalia, Y. N. & Guy, R. H. Passive skin penetration enhancement and its quantification in vitro. Eur. Pharm. Biopharm. 52, 103–112 (2001).il p 2. Pasumarty, S. M., Johnson, S. A., Watson, S. A. & Adams, M. J. Friction of the human finger pad: Influence of moisture, occlusion and velocity. Tribol. Lett. 44, 117–137 (2011). y 13. Hollins, M. & Risner, S. R. Evidence for the duplex theory of tactile texture perception. Percept. Psychophys. 62, 695–705 (2000).hfi 13. Hollins, M. & Risner, S. R. Evidence for the duplex theory of ta 4. Andre, T., Levesque, V., Hayward, V., Lefevre, P. & Thonnard, J.-L. Acknowledgementsh g The authors thank Simon Johnson of Unilever, R&D for his help with finger tribology. This study was funded by the FP7 Marie Curie Initial Training Network PROTOTOUCH, grant agreement No. 317100. It was also supported by the European Research Council (FP7) ERC Advanced Grant (PATCH) to V.H. (No. 247300). g The authors thank Simon Johnson of Unilever, R&D for his help with finger tribology. This study was funded by the FP7 Marie Curie Initial Training Network PROTOTOUCH, grant agreement No. 317100. It was also supported by the European Research Council (FP7) ERC Advanced Grant (PATCH) to V.H. (No. 247300). References Effect of skin hydration on the dynamics of fingertip gripping contact. J. R. Soc. Interface 8, 1574–1583 (2011). f 5. Terekhov, A. V. & Hayward, V. Minimal adhesion surface area in tangentially loaded digital contacts. J. Biomech. 44, 2508–2510 (2011).hi 6. Delhaye, B., Lefèvre, P. & Thonnard, J. Dynamics of fingertip contact during the onset of tangential slip. J. R. Soc. Interface 11 20140698 (2014).hhf 7. Gescheider, G. A., Thorpe, J. M., Goodarz, J. & Bolanowski, S. J. The effects of skin temperature on the detection and discrimination 14, 181–188 (1997).f 8. Bolanowski, S. J. & Verrillo, R. T. Temperature and criterion effects in a somatosensory subsystem: a neurophysiological and psychophysical study. J. Neurophysiol. 48, 836–855 (1982).hhl 19. Klöcker, A., Wiertlewski, M., Théate, V., Hayward, V. & Thonnard, J. L. Physical factors influencing pleasant touch during tactile exploration. PLoS One 8, doi: 10.1371/journal.pone.0079085 (2013).h p j p 0. Wiertlewski, M., Lozada, J. & Hayward, V. The spatial spectrum of tangential skin displacement can encode tactual texture. IEEE Trans. Robot. 27, 461–472 (2011).i 21. Wiertlewski, M., Hudin, C. & Hayward, V. On the 1/f noise and non-integer harmonic decay of the interaction of a finger slidi flat and sinusoidal surfaces. 2011 IEEE World Haptics Conf. WHC 2011 25–30, doi: 10.1109/WHC.2011.5945456 (2011). 21. Wiertlewski, M., Hudin, C. & Hayward, V. On the 1/f noise and non-integer harmonic decay of the interaction of a finger sliding on flat and sinusoidal surfaces 2011 IEEE World Haptics Conf WHC 2011 25 30 doi: 10 1109/WHC 2011 5945456 (2011) l p f 22. Adams, M. J. et al. Finger pad friction and its role in grip and touch. J. R. Soc. Interface 10, 20120467 (2013).fi g p g p f 23. Derler, S., Rossi, R. & Rotaru, G.-M. Understanding the variation of friction coefficients of human skin as a function of skin hydration and interfacial water films Proc Inst Mech Eng Part J J Eng Tribol 229 285 293 (2014) 23. Derler, S., Rossi, R. & Rotaru, G.-M. Understanding the variation of friction coefficients of human skin as a fun hydration and interfacial water films. Proc. Inst. Mech. Eng. Part J J. Eng. Tribol. 229, 285–293 (2014).̧f , , , , gfi ydration and interfacial water films. Proc. Inst. Mech. Eng. Part J J. Eng. Tribol. 229, 285–293 (2014).̧f i 24. Dinç, O. S., Ettles, C. M., Calabrese, S. J. & Scarton, H. A. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Gueorguiev, D. et al. Touch uses frictional cues to discriminate flat materials. Sci. Rep. 6, 25553; doi: 10.1038/srep25553 (2016). How to cite this article: Gueorguiev, D. et al. Touch uses frictional cues to discriminate flat materials. Sci. Rep. , 25553; doi: 10.1038/srep25553 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W2025870962	https://zenodo.org/records/2320836/files/article.pdf	German	null	Untersuchung der Turbulenz beim Durchströmen von Wasser und Quecksilber durch spiralförmig gewundene Kapillaren	Annalen der Physik	1,913	public-domain	8,089	1) Auszug aus der Wiirzburger Dissertation. 614 614 1) E. Griineisen, Wissenschaftliche Abhandlungen der Phys.- Techn. Reichsanstalt, 4, Heft 2, p. 179. 2) 0. Reynolds, On the dynamical theory of incompressible vis- cous fluids and the determination of the criterion. London. Phil. Trans. (A 186). 1895. 5) 0. Reynolds, An experimental investigation of the circumstmce, which determine whether the motion of water shall be direct or sinuous and of the law of resistance in parallel channels. London. Phil. Trans. 174. 1883. 5) 0. Reynolds, An experimental investigation of the circumstmce, which determine whether the motion of water shall be direct or sinuous and of the law of resistance in parallel channels. London. Phil. Trans. 174. 1883. 6. Umter.uuchuny d er Turbu 1 ens beim DurchstrSmen von Wauser uncl Quecksilber durch apiralfSrmig gewundene Kapillaren I); uon Gottfried Lechner. 6. Umter.uuchuny d er Turbu 1 ens beim DurchstrSmen von Wauser uncl Quecksilber durch apiralfSrmig gewundene Kapillaren I); uon Gottfried Lechner. 6. Umter.uuchuny d er Turbu 1 ens beim DurchstrSmen von Wauser uncl Quecksilber durch apiralfSrmig gewundene Kapillaren I); uon Gottfried Lechner. Das von Poiseuille fur den DurchfluS von Flussig- keiten durch gerade Kapillaren aufgestellte Qesetz lautet be- kanntlich: 72 v = --r4.t(pa-pp,). 8 7 1 Fur die Geschwindigkeit, die in einer Entfernung 9 von der Kapillarenachso vorhanden ist, gilt ferner : In diesen Formeln bedeuten: Y das durch das Eode ausgetretene Fliissigkeits- volumen, T den Radius der Rohre, T den Radius der Rohre, p , den Druck am Anfang der Rohre, p , den Druck am Anfang der Rohre, P, 17 91 11 Ende JY 11 P, 17 91 11 Ende JY 11 t die Ausstromungszeit, , t die Ausstromungszeit, , q ,) Reibungskonstante. q ,) Reibungskonstante. Alle vorkommenden GriiSen sind im C.G.S.-System ge- messen. Die strenge Giiltigkeit des Gesetzes setzt voraus, da@ ein Stromuogszustand vorhanden ist, bei dem die Stromlinien parallel zur Achse der Kapillare sind, eine Forderung. die urn so geoauer erreicht wird, je kleiner die Geschwindigkeiten sind, welche die Flussigkeit beim Durchstromen besitzt. Es macht sich in diesem Falle der EinfluB der Enden der Rohre wenig 615 Untersuchung det Turbulenz usw. bemerkbar. Letzterer l) namlich besteht darin, daS an den Kapillarenenden eine Abweichung von der parabolischen Ge- achwindigkeitsverteilung auftritt, wie sie im Innern an jeder Stelle des Querschnitts vorhanden ist. Bei zunehmender Ge- schwindigkeit zieht sich nun diese anormale Geschwindigkeits- verteilung allmahlich immer weiter in das Innere der Rohre hinein und bringt so Abwveichungen vom Poiseuilleschen Gesetz hervor. Bei weiterer Erhohung der Geschwindigkeit fand nun Osborne Reynolds, daS der dem obigen Gesetze entsprechende Stromungszustsnd aufhart stabil zu sein, indem namlich von einer bestimmten Geschwindigkeit an, die er ,,kritische Geschwindigkeit" nennt , wirbelformige Bewegungen der Fliissigkeitsteilchen zutage treten. R ey n o 1 d s hat dieses Auftreten von Wirbeln theoretisch 2, sowohl als experimentell 3, untersucht nnd fand als Ausdruck fur diese Grenzgeschwindig- keit beim Ubergang zum Turbulenzzustand: Hierin bedeuten : x eine numerische 'Konstante, deren Wert fur kreis- formige Querschnitte zwischen 1900 und 2000 schwankt, U, die mittlere Geschwindigkeit, U, die mittlere Geschwindigkeit, q ,, Reibungskonstante, g ,, Dichte, d den Durchmesser der Kapillare. 2) 0. Reynolds, On the dynamical theory of incompressible vis- cous fluids and the determination of the criterion. London. Phil. Trans. (A 186). 1895. 1) E. Griineisen, Wissenschaftliche Abhandlungen der Phys.- Techn. Reichsanstalt, 4, Heft 2, p. 179. d den Durchmesser der Kapillare. d den Durchmesser der Kapillare. Die mittlere Geschwindigkeit ergibt sich aus Formel (2) wenn man das Mittel nimmt aus der zu p = o und p = r ge- horigen Geschwindigkeiten, d. h. sie ist gleich der halben in G. Lechner. 616 der Richtung der Achse vorhandenen Geschwindigkeit; man findet also fiir ihren Wert: (4) Fiir den kritischen Druck erhalt man dann : Fiir den kritischen Druck erhalt man dann : n q l u, - 3 2 ' 1 ' 1 . ~ re e . 8 * P = A (5) Es war nun von Interesse die experimentellen Unter- suchungen uber das Auftreten dieses Turbulenzzustandes auf Kapillaren auszudehnen, die von der geradlinigen Form ab- weichen. Was nun das Poiseuillesche Gesetz im Falle von gebogenen Kapillaren betrifft, so haben schon friiher ver- schiedene Autoren l) darauf hingewiesen , da6 bereits bei niedrigen Drucken wesentliche Abweichungen eintreten. In welcher Weise aber bei gekriimmten Kapillaren das Reynolds- sche Kriterium beeinflu6t wird, dariiber sind meines Wiusens noch keine aufschlut3gebenden Versuche unternommen worden. g Ich habe daher von Herrn W. Wien die Aufgabe gestellt bekommen zu untersuchen , welchen Einfluf3 die Kriimmung von engen langen Kapillaren auf das Eintreten des Turbulenz- zustandes hat. Zu diesem Zwecke habe ich spiralformig ge- wundene Kapillaren verwendet und folgende Fragen zu be- antworten gesucht: g Wie iindert sich das Reynoldssche Kriterium: a) Bei Vermehrung der Windungen? b) Bei Anderung des Kapillarenradius? Zur Beantwortung dieser Fragen habe ich die Versuche mit Wasser und Quecksilber durchgefuhrt. 1) Gotthilf Haffner, Erlanger Dissertation p. 16.1903; H. Egger, 2) Wilhelm Ruckes, Wiirzburger Dissertation p. 12, 13. 1908. Ann. d. Phys. 27. p. 835. 1098. 2) Wilhelm Ruckes, Wiirzburger Dissertation p. 12, 13. 1908. y 1) Gotthilf Haffner, Erlanger Dissertation p. 16.1903; H. Egger, Ann. d. Phys. 27. p. 835. 1098. 1) Es sei hier erwiihnt, dab die oben beschriebene Anordnung zum Teil von E. Schnetzler herruhrte, wie el. sie in seiner in den Verhandl. d. Deutsch. Physik. Ges. p. 817. 1910 veroffentlichten Arbeit benutzte. A l d Ph lk IV F l 42 40 y p Anoalen der Physlk. IV. Folge. 42. 40 Vereuahsanordnung. Bei den in vorliegender Arbeit ausgefuhrten Versuchen wurde zum gro6ten Teile die im physikalischen Institute vor- handene Hochdruckanlage a) beniitzt, indem letztere so er- Uiit h d T bd 617 Uiitersuchung der Turbdenz usw. 617 weitert wurde, daB auch Messungen mit Drucken von einigen Zentimetern Quecksilberhohe ausgefuhrt werden konnte. Die - ganze Druckanlage sei an der Hand der Fig. 1 im -- : I ci- folgenden kurz skizziert. : I ci- K Fig. 1. Mit Hilfe eines Stufen- kompressors 21, der durch einen Gasmotor G ver- mittelst der Transmission T in Betrieb gesetzt wurde, konnte Luft aus dem Freien in die drei ubereinander- liegenden Reservoire R,, R,, R3, gepumpt worden. Das Ventil 7, schlo6 die drei Luftraume gegen den Kom- pressor ab, wenn die Luft auf den Maximaldrdck von 200 Atmospharen gebracht K Fig. 1. Dieser Druck konnte an dem Metallmanometer M abgelesen werden. Zwischen dem Leitungsrohr L, und dem Verbindungsrohr der drei Reservoire war ein weiteres Ventil 7, angebracht; ebenso waren L, und A, durch ein Ventil V, voneinander getrennt. Der Zweck dieser beiden letzten Ventile wird dann bei der Beschreibung der eigentlichen Versuchsanordnung naher erlautert werden. Fig. 1. Die eigentliche Versuchsanordnung l) war durch das Leitungs- rohr L, mit der Hochdruckanlage in Verbindung gesetzt. S. Fig. 2. Es fuhrte namlich L, zu den beiden Eisenbomben B, und B,. In diese waren luftdicht die Metallrohrchen r,, T,, r3 eingelotet, deren Durchmesser im Verhaltnis zu dem der Eisenbomben sehr klein war. Das Rohrchen rg, miindete in die Kisenflasche E. In letztere war ein T-formiges Metallrohr ein- geschraubt. Der eine Arm 1 diente zum Anbringen der zu untersuchenden Kapillaren, wahrend der andere zu den Mano- metern fuhrte, mit denen der Druck des durch die Kapillaren 40 G. Lechner. 618 stromenden Wassers gemessen werden konnte. Arm 3, der wahrend der Messungen geschlossen blieb, diente als Zuleitung zur Fiillung der Bomben mit Wasser. Das zweite Ende der Kapillrtre war in ein weites Rohr m, gekittet, in das ein Quecksilberthermometer eintauchte , um die Temperatur deu Fig. 2. Fig. 2. durchstrijmenden Wassers messen zu konnen. m, verzweigte sich mit Hilfe eines gabelformigen Glasrohres in die Leitungen W, und W2, welche durch einen Schnapphahn S zu den Aus- flu6gefaBen fiihrten. Die Einrichtung des Schnapphahns werde ich an einer spateren Stelle beschreiben. durchstrijmenden Wassers messen zu konnen. Vereuahsanordnung. m, verzweigte sich mit Hilfe eines gabelformigen Glasrohres in die Leitungen W, und W2, welche durch einen Schnapphahn S zu den Aus- flu6gefaBen fiihrten. Die Einrichtung des Schnapphahns werde ich an einer spateren Stelle beschreiben. Bei allen Versuchen wurde destilliertee Wasser verwendet, das in die Bomben B, und b', gebracht wurde. Mit Hilfe des oben erwahnten Ventiis 5 konnte aus den Reservoiren beliebig vie1 komprimierte Luft entnommen werden und der jeweilige Druck am Manometer M abgelesen werden. Um nun von dieser Druckluft zur Erzeugung von niedrigen Drucken kleinere Volumina verwenden zu konnen , diente das Ventil V4 (s. Fig. l), das mit einer Hebeliibertragung versehen war und beliebig kleine Volumina in die Leitung L, und dann in die Bomben gelangen lieB. Infolge des Druckes, den die Luft auf das Wasser in der Bombe B, ausiibte, wurde dieses durch die Rohrchen r,, r,, rg in die Eisen- flasche E uud in die Kapillaren getrieben. Durch den 619 Untetsuchung der firbulenz usw. Arm 2 floB es zu den Manometern M, und M,, welche ebenso wie die Kapillaren beim Fullen der Bomben entfernt wurden. M, war ein offenes Hg-Manometer. An den Arm 2 war ein Windkessel R angeschraubt. Durch den Gummischlauch G war er mit einem Schenkel von M, verbunden. Durch das in den Windkessel einstromende Wasser wurde die Luft in ihm komprimiert und der Druck an Ma abgelesen. Vor den Messungen wurden die Schraubchen b, und b, an K ent- fernt, um in K den auBeren Luftdruck herzustellen. Mit M, konnten Drucke bis zu 3 Atmospharen gemessen werden. Bis zu Drucken von 30 Atmospharen diente dann das ge- schlossene Wassermanometer M, , welches folgendermaBen ein- gerichtet war. Einem moglichst widerstandsfahigen , oben ge- schlossenen Glasrohr, von der Lange I = 240 cm und dem inneren Durchmesser d = 2,5 cm war eine aus Fig. 3 zu ersehende Form gegeben worden. Mit Hilfe von Siegellack war es in ein Messingzylinderchen m eingekittet. Durch a konnte Wasser hereinstromen. In das Manometer- rohr reichte bis zu einer bestimmten Stelle ein Messingrohrchen d von 2 mm Durchmesser. Durch Anbringen der Schraube f wurde m geschlossen. An der Hinterseite des Glas- rohres war ein PapiermaSstab angeklebt, dessen Nullpunkt genau mit dem Ende von d in einer horizontalen Ebene lag. Das Volumen des Manometerrohres vom Nullpunkt bis zu einem beliebigen Skalenteil des MaBstabes war durch Auswagen mit Quecksilber bestimmt punkt der Teilnng eingestellt wurde, was leicht worden. Vereuahsanordnung. Bei den Messungen wurde nun so ver- fahren, daB bei Beginn derselben die Schraube f entfernt und das Wasser genau auf den Null- zu erreichen war, da das uberfliissige Wasser durch d ab0ieBen konnte. Gleichzeitig hatte das Rohrchen d den Zweck im Manometer den auBeren Luftdruck herzustellen , so daB, wenn durch die Schraube f die Verbindung mit der au6eren Luft abgesperrt war, dem Gesamtvolumen vom Nullpunkt an der auBere Luftdruck entsprach. Man konnte also Ilr Fig. 3. 41 Fig. 3. Fig. 3. 41 41 620 G. Lechner. pu=const. berechnen. Da das zu einem Skalenteil des Ma6- stabes gehorige Volamen bekannt war, so lie6 sich aus voraus- gehender Gleichung der zugehorige Druck sofort berechnen. Praktisch wurde diese Druckbestimmung durchgefuhrt, indem an mehreren Stellen, hauptsachlich an den Verengungsstellen, der zu einem Teilstrich gehorige Druck mit Hilfe obiger Beziehung bestimmt und dann Druck und Skalenteil als Ordinaten und Ab- szissen aufgetragen wurde. Fur einen beliebigen Skalenteil wurde der Druck aus der so erhaltenen Kurve entnommen. Falls zu viel Luft in die Bomben eingestromt war, so wurde durch Drehen der Kreisscheibe s, das Ventil g (8. Fig. 2) geoffnet und da- durch ein Teil der Luft zum Entweichen gebracht. Zur rascheren Handhabung wurde um die Kreisscheibe ein Riemen gelegt und mit Hilfe der Raderchen g der Riemenleitung eine aus Fig. 2 zu ersehende Form gegeben. Durch Anziehen an den Schniiren 1 und 2 konnte g geoffnet und geschlossen werden. pu=const. berechnen. Da das zu einem Skalenteil des Ma6- stabes gehorige Volamen bekannt war, so lie6 sich aus voraus- gehender Gleichung der zugehorige Druck sofort berechnen. Praktisch wurde diese Druckbestimmung durchgefuhrt, indem an mehreren Stellen, hauptsachlich an den Verengungsstellen, der zu einem Teilstrich gehorige Druck mit Hilfe obiger Beziehung bestimmt und dann Druck und Skalenteil als Ordinaten und Ab- szissen aufgetragen wurde. Fur einen beliebigen Skalenteil wurde der Druck aus der so erhaltenen Kurve entnommen. Falls zu viel Luft in die Bomben eingestromt war, so wurde durch Drehen der Kreisscheibe s, das Ventil g (8. Fig. 2) geoffnet und da- durch ein Teil der Luft zum Entweichen gebracht. Zur rascheren Handhabung wurde um die Kreisscheibe ein Riemen gelegt und mit Hilfe der Raderchen g der Riemenleitung eine aus Fig. 2 zu ersehende Form gegeben. Durch Anziehen an den Schniiren 1 und 2 konnte g geoffnet und geschlossen werden. l) E. S c h n e t z 1 e r, Verhandlungen der Deutsch. Physik. Gesell- schaft, Berlin 1910. p. 817. Vereuahsanordnung. Da namlich infolge des Ausstromens des Wassers aus den Bomben die iiber ihm lagernde Luftmenge sich auf einen gro6eren h u m ausbreitete, so blieb der Druck nicht konstant und bei Drucken von 100 cm Hg betrug der Unter- schied zwischen Anfangs- und Enddruck bereits mehr als 30 cm. Durch Regulieren des Ventils T4 konnte man zwar Luft in die Bomben nachstromen lassen, aber zu exakteren Korrektionen war dieses Verfahren ungenugend. Ich habe daher zwischen L, und den Bomben ein sehr feines Nadel- ventil N eingescbaltet. Die Druckschwankungen wurden dann derart ausgeglichen , daB Ventil 7, wahrend der Messungen passend geoffnet blieb, so daB ein verhaltnisma6ig feiner Lufb strom zu N gelangen konnte. Durch Regulieren von N gelang es mir dam, die Drucke bei beiden Manometern bis auf 1 mm konstant zu erhalten. Es moge an dieser Stelle der Zweck der beiden Eisenbomben Bl und B2 erwahnt werden: ,,Das1) unter hohen Druck stehende Wasser absorbierte so viel Luft, daB es fur die Untersuchungen nicht zu gebrauchen war. Es wurden deshalb zwei Kohlensaurebomben hintereinander ge- Untersuchung der Turbulenz mu). 62 1 schaltet und durch die engen Verbindungsrohrchen verhindert, da6 die absorbierte Luft in die zweite Flasche diffundierte." Was die Beobachtung des ausgetretenen Fliissigkeits- volumen bet& sowie der dazu notwendigen Zeit, so mochte ich mit einer Erklarung dieser Beobachtungsmethoden die Beschreibung des Schnapphahns verbinden. Zwischen zwei festen Messingplatten m, und ma (8. Fig. 4) von der Dicke d = 4 mm konnte ein weiterer Messingstreifen m, (Lange: 20 cm, Breite: 4 cm, Dicke: 2,5 mm) durch Einfetten mit Talg sich mit sehr geringer Reibung bewegen. m, und ma waren mit zwei kreisftirmigen, 3 cm voneinander entfernten Aus- schnitten vom Durchmesser d = 2 cm versehen. Letztere lagen Fig. 4. genau einander gegeniiber. Die Ausschnitte von ml waren mit der gabelf6rmig verzweigten Zuleitung verbunden, die von m2 rnit den beiden AusfluBleitungen. Der bewegliche Streifen m, besa6 ebenfalls zwei kreisfdrmige Ausschnitte von der gleichen QroSe. Die Stellung von m, war stets eine solche, daB zwei gegeniiberliegende Ausscbnitte von m, und m, durch den gleich- gro6en von m, verbunden waren, wahrend die beiden anderen durch den Streifen zwischen den Ausschnitten von m, getrennt waren. Es konnte also stets durch eine Leitung Wasser aus- stromen. Nehmen wir an, in 1 sei die Verbindung hergestellt. Vereuahsanordnung. Durch Verschieben von T nach rechts wurden die Federn .PI gespannt, durch Ziehen an der Schnur 2 wurde der Hebel H, gelost, m, gab dann der gespannten Feder Fl nach, unterbrach die Offnung bei 1 und stellte die Verbindung durch 2 her, so daB also jetzt durch 2 Wasser hindurchstromen konnte. Gleich- zeitig w d e durch Ziehen an der Schnur 2 eine Stoppuhr in Tatigkeit gesetzt. Durch Verschieben von T nach links wurden jetzt die Federn F, gespannt, durch Ziehen an der Schnur 1 der Hebel Hl gelost. m, gab der gespannten Feder Fig. 4. Fig. 4. Fig. 4. genau einander gegeniiber. Die Ausschnitte von ml waren mit der gabelf6rmig verzweigten Zuleitung verbunden, die von m2 rnit den beiden AusfluBleitungen. Der bewegliche Streifen m, besa6 ebenfalls zwei kreisfdrmige Ausschnitte von der gleichen QroSe. Die Stellung von m, war stets eine solche, daB zwei gegeniiberliegende Ausscbnitte von m, und m, durch den gleich- gro6en von m, verbunden waren, wahrend die beiden anderen durch den Streifen zwischen den Ausschnitten von m, getrennt waren. Es konnte also stets durch eine Leitung Wasser aus- stromen. Nehmen wir an, in 1 sei die Verbindung hergestellt. Durch Verschieben von T nach rechts wurden die Federn .PI gespannt, durch Ziehen an der Schnur 2 wurde der Hebel H, gelost, m, gab dann der gespannten Feder Fl nach, unterbrach die Offnung bei 1 und stellte die Verbindung durch 2 her, so daB also jetzt durch 2 Wasser hindurchstromen konnte. Gleich- zeitig w d e durch Ziehen an der Schnur 2 eine Stoppuhr in Tatigkeit gesetzt. Durch Verschieben von T nach links wurden jetzt die Federn F, gespannt, durch Ziehen an der Schnur 1 der Hebel Hl gelost. m, gab der gespannten Feder 622 G. Lechner. nach, unterbrach jetzt wieder die Offnung 2 und stellte die durch 1 her. Gleichzeitig wurde durch Anziehen an 1 die Stoppuhr zum Stillstand gebracht. Man konnte so genau die Zeit featstellen, wahrend welcher das Wasser durch eine Offnung stromte. Vor den Messungen wurde das Wasser abwechselnd durch beide Leitungen geschickt, bis es aus den AusfluS- gefaBen A, und A, ausstromte. Letztere umschlossen dicht anliegend schnabelf6rmige aus Aluminium gefertigte Schalen, aus denen es dann in die Auffangegefa6e strdmte. Diesel) Schnapphahneinrichtung hnktionierte sehr gut. In dem Augen- blick, in welchem die Ausstromung durch A, unterbrochen wurde, trat die durch A, ein. 1) E. Schnetzler, Verhandlnngen der Deutschen, Physik. Ge- sellschaft, Berlin p. 817. 1910. 1) Siehe Wilhelm Ruckee, Wiirzburger Dissertation p. 15 u. 16. 2) Ebenda p. 16. Vereuahsanordnung. Indem ich am Anfang der Messungen Wasser unter hohem Druck dnrchstromen lieB, gelang es mir, die Luft- blasen in der Kapillare und den anschliegenden Leitungen zu entfernen. Was die Bestimmung der Durchmesser betrifft, so habe ich sie tttjls mikroskopisch, teils durch Auswagen, teils aber auch durch Durchstromungsversuche nach dem Poiseuille- schen Gesetze bestimmt. Die Langen wurden bei geraden Kapillaren mit einem BandmaB gemessen, bei gewundenen habe ich einen sehr feinen Faden mit Klebwachs an den einzelnen Windungen befestigt und nachtriiglich dessen Lange bestimmt. Die Bestimmung des Kriimmungshalbmessers geschah in der Weise, daB an zwei diametral gegeniiberliegenden Punkten einer Windung ein dunner Faden befestigt wurde, dessen Lange mit einem MaBstab gemessen wurde. Die ge- wundenen Kapillaren bezog ich von C. Kramer in Freiburg; sie hatten folgende Form /”b””d. Die Entfernung zwischen zwei Windungen betrug hier im Mittel ungefhhr 1 cm B war an den Arm 2 angeschraubt (8. Fig. 2). moglichst dicht waren. Die Ventile hatten eine aus Fig. 6 zu ersehende Form. l) Diese Dichtungen geschahen durch Aluminiumscheibchen, die sich im Verlauf der Messungen als sehr haltbar und dicht bewiesen. Die Glaskapillaren, welche an beiden Enden glatt abgeschnitten waren, wurden mit Siegel- lack in eine Messingbuchse B eingekittet (8. Fig. 7). Diese2) Art der Kittung hat sich auch bei hohen Drucken bewahrt, 80 daB nicht ein einziges Ma1 eine Kapillare herausgeschleudert wurde. Die Kapillaren selbst waren wahrend der Messungen in ein Tem- peraturbad eingetaucht. Ich habe auch Messungen vor- genommen ohne ein Bad zu benutzen und dabei gefunden, daB nicht die geringste inderung in bezug auf die Temperatur des durchgestromten Wassers eintrat. Ich konnte daher, ohne einen Fehler zu begehen , das Temperaturbad weglassen, namentlich bei kompliziert gewundenen Rohren. Weitere Sorgfalt war auf die in den Leitungen auftretenden Luftblasen zu verwenden. Indem ich am Anfang der Messungen Wasser unter hohem Druck dnrchstromen lieB, gelang es mir, die Luft- blasen in der Kapillare und den anschliegenden Leitungen zu entfernen. Was die Bestimmung der Durchmesser betrifft, so habe ich sie tttjls mikroskopisch, teils durch Auswagen, teils aber auch durch Durchstromungsversuche nach dem Poiseuille- schen Gesetze bestimmt. Die Langen wurden bei geraden Kapillaren mit einem BandmaB gemessen, bei gewundenen habe ich einen sehr feinen Faden mit Klebwachs an den einzelnen Windungen befestigt und nachtriiglich dessen Lange bestimmt. Vereuahsanordnung. Das aus den Schalen kommende Wasser wurde bei kleinen Volumina in gewohnlichen bis 100 ccm reichenden Glasmensuren aufgefangen. Bei groSeren Volumina wurde folgende Methode angewendet (s. Fig. 5). Dem I uKapillare i I Fig. 7. I uKapillare Alumin Scheibch Fig. 5. Fig. 6. i I Fig. 7. Schwimmer S wurde durch ein gleichgroBes Gewicht G das Gleichgewicht gehalten. An G befand sich eine feine Spitze, die vor einem kleinen Magstab m sich bewegen konnte. Das zu einigen Skalenteilen gehorige Volumen war gemessen und Volumen und Teilstrich graphisch dargestellt worden. Aus der so erhaltenen Kurve konnte nun das zu einem Teilstrich gehorige Volumen entnommen werden. Fig. 5. Alumin Scheibch Fig. 6. Fig. 6. Fig. 5. Fig. 7. Schwimmer S wurde durch ein gleichgroBes Gewicht G das Gleichgewicht gehalten. An G befand sich eine feine Spitze, die vor einem kleinen Magstab m sich bewegen konnte. Das zu einigen Skalenteilen gehorige Volumen war gemessen und Volumen und Teilstrich graphisch dargestellt worden. Aus der so erhaltenen Kurve konnte nun das zu einem Teilstrich gehorige Volumen entnommen werden. Schwimmer S wurde durch ein gleichgroBes Gewicht G das Gleichgewicht gehalten. An G befand sich eine feine Spitze, die vor einem kleinen Magstab m sich bewegen konnte. Das zu einigen Skalenteilen gehorige Volumen war gemessen und Volumen und Teilstrich graphisch dargestellt worden. Aus der so erhaltenen Kurve konnte nun das zu einem Teilstrich gehorige Volumen entnommen werden. Bei den einzelnen Messungen war in erster Linie darauf zu sehen, daS die Ventile und Kittstellen der Kapillaren lintersucfiunq der Turbulenz usw. 623 moglichst dicht waren. Die Ventile hatten eine aus Fig. 6 zu ersehende Form. l) Diese Dichtungen geschahen durch Aluminiumscheibchen, die sich im Verlauf der Messungen als sehr haltbar und dicht bewiesen. Die Glaskapillaren, welche an beiden Enden glatt abgeschnitten waren, wurden mit Siegel- lack in eine Messingbuchse B eingekittet (8. Fig. 7). Diese2) Art der Kittung hat sich auch bei hohen Drucken bewahrt, 80 daB nicht ein einziges Ma1 eine Kapillare herausgeschleudert wurde. Die Kapillaren selbst waren wahrend der Messungen in ein Tem- peraturbad eingetaucht. Ich habe auch Messungen vor- genommen ohne ein Bad zu benutzen und dabei gefunden, daB nicht die geringste inderung in bezug auf die Temperatur des durchgestromten Wassers eintrat. Ich konnte daher, ohne einen Fehler zu begehen , das Temperaturbad weglassen, namentlich bei kompliziert gewundenen Rohren. Weitere Sorgfalt war auf die in den Leitungen auftretenden Luftblasen zu verwenden. 2) Ebenda p. 16. Vereuahsanordnung. Die Bestimmung des Kriimmungshalbmessers geschah in der Weise, daB an zwei diametral gegeniiberliegenden Punkten einer Windung ein dunner Faden befestigt wurde, dessen Lange mit einem MaBstab gemessen wurde. Die ge- wundenen Kapillaren bezog ich von C. Kramer in Freiburg; sie hatten folgende Form /”b””d. Die Entfernung zwischen zwei Windungen betrug hier im Mittel ungefhhr 1 cm. B war an den Arm 2 angeschraubt (8. Fig. 2). 624 G. Lechner. Vorversuche. Die in diesem Abschnitt durchgefuhrten Vereuche ge- schahen in der Weise, daS ich das ausgetretene Fliissigkeits- volumen sowie den zugehorigen Druck bestimmte und dann graphisch darstellte. Das dem Reynoldsschen Kriterium entsprechende Volumen, bzw. den Druck, erhielt ich sls scharfen Knick in der geradlinig ansteigenden Druckvolumen- kurve. Dabei nahm ich zueret eine gerade Kapillare, deren Dimensionen nach obigen Angaben genau bestimmt waren. Hierauf lie6 ich die Rohre mit Windungen versehen und be- stimmte wieder Druck- und Volumenkurve. Die so erhaltenen Kurven konnten nun miteinander verglichen werden. Da im voraus iiber die GroSe einer Abweichung bzw. einer Ver- schiebung des Knickes nichts Positives vorherzusagen war, ao war es vorteilhaft, eine Kapillare mit moglichst vielen Win- dungen und verhaltnisma6ig kleinem Krummungshalbmesser zu nehmen. Auf diese Weise habe ich folgende sechs Kapillaren gemessen. Ihre Gro6enverhaltnisse, sowie Windungszahl und Krummungshalbmesser sind in nachfolgender Tabelle an- gegeben. Durchmesser in cm ~ -. __ Liinge Kriimrnung in cm in cm Rapillare I1 I11 I IV V VI - ~ _ _ _ _ 0,112 0,116 157,7 795 157,7 7,5 Wind.-Zahl ____ ____ 14 14 14 5 5 5 0,111 I 157,4 795 Die Kurventafeln I-VI zeigen die erhaltenen Druckvolumen- kurven. Als Abszisse ist immer der halbe Druck aufgetragen, als Ordinate das zugehiirige Volumen. Das Druckintervall er- streckte sich hierbei, da samtliche kritische Drucke bei mittleren Werten liegen, von 2 cm bis 2l/, Atmospharen. In der Tab. 2 sei als Beispiel der Beobachtungen die einzelnen Werte fiir die ge- rade ale fur die gewundene Form der Rohre 2 wiedergegeben. 625 U7itersuchun.q der Turbulenz usw. Bevor ich auf eine nahere Diskussion der Abweichungen eingehe, welche schon bei ganz niedrigen Quecksilberdrucken auftreten, scheint es mir vorteilhaft zu sein, das Druckintervall sowohl aufwarts zu hoheren Drucken, als abwarts zu niedrigen Taf. I. Taf. 11. Taf. I. Taf. 11. 626 G. Lecher. 150 100 50 0 40 Taf. 111. 150 100 50 0 40 Taf. 111. Untersuchun.q der Turbulenz usto. 627 628 G. Acchner. Tabelle 2. Gerade Druck in ccm 10 20 30 40 50 60 70 - - - - Volumen in ccm 30,9 61,9 92,O 111,2 121,o 130,O 138,O _____ . ____ - - - - Gebogen in ccm in ccm -~ --- lo 5 20 25 30 35 40 50 67,O 60 76,8 70 86,1 628 G. Acchner. G. Acchner. Tabelle 2. Tabelle 2. zu erweitern. Zu diesen Messungen verwendete ich die Ka- pillare 2. Vorversuche. Ich ging zunachst bis zu Drucken von 20 Atmo- sphiiren. Es zeigte sich aber nichts Neues. Die beiden Tur- bulenzkurven, niimlich die der geraden und gewundenen Ka- pillare, laufen parallel zueinander. Urn ferner bei ganz Fig. 8. Fig. 8. niedrigen Drucken arbeiten zu khnen, gebrauchte ich die in Fig. 8 dargestellte Anordnung, deren Handhabung ohne weiteree aus der Figur zu ersehen ist. Fig. 8. Fig. 8. niedrigen Drucken arbeiten zu khnen, gebrauchte ich die in Fig. 8 dargestellte Anordnung, deren Handhabung ohne weiteree aus der Figur zu ersehen ist. 629 Ehtersuchung der fitbulenz usw. Ich verwendete bei diesen Messungen Drucke von 2 bis 57 cm H,O. Nach Reduktion des Volumens auf das in einer Minute durchgestromte, erhielt ich nachfolgende Druckvolumen- kurve. Die der geraden Kapillare entsprechende Kurve habe ich nicht gezeichnet, sondern aus den bekannten Dimensionen berechnet. Als Abszisse ist hier das Volumen pro Minute, als Ordinate wieder der halbe Druck (vgl. Fig. 9). so 28 26 24 22 20 18 16 I4 12 10 8 6 b." Volumen in cm 2 \ 0 1 2 5 4 5 Fig. 9. Fig. 9. Wenn ich nun zur Betrachtung der bedeutenden Ab- weichungen ubergehe, so liegt zunachst die Vermutnng nahe, da8 sie von einer beim Winden eingetretenen Querschnitts- deformation herriihren konnten. Urn hieriiber Klarheit zu ver- schaffen, habe ich die gewundenen Eapillaren an mehreren Richtung I 0,105 0,102 ,, I1 1 0,105 0,102 ,, 111 , 0,108 0,102 0,104 0,105 0,104 Tabelle 3. Tabelle 3. 1) Vgl. Griitz, Schlomilchs Zeitachrift fiur Mathematik %. p. 316. 1579. Tabelle 3. 630 G. Lecher. Stellen abgesclinitten und an jeder Stelle in drei um einen Winkel von 120 O auseinander liegenden Richtungen den Durch- messer mikroskopisch bestimmt. Far eine Stelle am Anfang, in der Mitte und am Ende seien in vorstehender Tabelle 3 die Resultate angegeben. Aus diesen Werten sieht man ohne weiteres, daB sie nicht imstande sind, die Abweichungen zu erklaren, die schon bei ganz niedrigen Drucken auftreten. Ich habe auch gerade Ka- pillaren an verschiedenen Stellen in bezug auf die Gleichheit ihres Querschnittes untersucht und die namlichen Verande- rungen des Durchmessers gefunden.3 Aus dem Poiseuille- schen Gesetze ergab sich fiir den Durchmesser in Rohre 2 d = 0,106 cm. Betrachten wir die Abweichungen, so fallt vor allem bei samtlichen Kurven von 1-6 das Fehlen des Knickes auf. Es la& sich aus dieser Tatsache entschieden der SchluB ziehen, da6 kein unstetiger , sondern ein allmahlicher Ubergang von der Poiseuilleschen Stromung zum Turbulenzzustand statt- findet. Ferner ist beachtenswert, daB die Kurven der ge- raden und gekriimmten Kapillare bei hoheren Drucken parallel werden und zwar liegt die Stelle, an welcher bei den gewun- denen Kapillaren der Parallelismus beginnt, bei kleineren Volumina. Es scheinen also diese Versuche dafiir zu sprechen, daB durch Windungen der Eintritt der Turbulenzstromung be- schleunigt wird. Genaueren AufschluB dariiber bringen nun die folgenden Versuche. Hauptvereuche. Nachdem die Vorversuche wichtige Aufschliisse iiber den Eintritt der Turbulenzstromung (insbesondere der Abhangigkeit vom Kapillarenradius) geliefert hatten, habe ich in den fol- genden Messungen in erster Linie den EinfluB der Windungs- zahl gepriift. Zu diesem Zwecke habe ich ein und dieselbe Rohre zuerst mit einer Windung versehen lamen und die Beobachtungen angestellt, dann habe ich bei 2, 4, 6, 8 Win- Untersuchung der Turbulenz usw. 631 dungen die Messungen ausgefiihrt. Mehr als acht Windungen konnte ich nicht nehmen, da bei dem gewahlten Kriimmungs- halbmesser die Lange der Rohre zu weiteren Windungen nicht ausreichte. Die erhaltenen Druckvolumenkurven sind in der Tafel VII (vgl. p. 632) dargestellt und zwar der halbe Druck als Abszisse, das in einer Minute ausgestromte Volumen als Ordi- nate. Die der geraden Kapillare entaprechende Kurve wurde nicht beobachtet, sondern aus bekannten Dimensionen der Rohre berechnet. Der aus dem vorausgehenden Abschnitt gefundene Parallelismus dieser Turbulenzkurven ergab die Richtung von 1 b. Die Lange der Kapillare betrug 1 = 157,6 cm, der Kapillaren- durchmesser 2 Q = 0,125 cm , der Kriimmungshalbmesser 2 r = 4,5 cm. Vergleichen wir die Kurven 2-6, so fallt vor allem wieder der Parallelismus der Turbulenzzweige auf. Ferner sind die Abstande, d. h. die Abweichungen der Volumina bei ein und demselben Drucke sehr nahe einander gleich, namentlich die der parallelen Aste, so daB man also eine direkte Proportio- nalitat zwischen Windungszahl und Abweichung annehmen darf, sicherlich, wenn es sich um so wenige Windungen handelt, wie in den gezeichneten Kurven. Ich mochte hier gleich er- wahnen, daB die einzelnen Windungen nicht ganz gleichmaBig waren und Unterschiede in den Durchmessern der Windungen bis zu 5 mm vorkommen. Ferner waren die einzelnen Kriim- mungen nicht exakt kreisfdrmig. Auf diese Tatsache glaube ich zuruckfuhren zu durfen, daB die Proportionalitit nicht vollstiindig genau erhalteu wurde. Doch scheint sich dieser EinfluB bei den der Kurve l a entsprechenden Teilen mehr geltend zu machen, als bei denen, die zu l b gehoren. Das Fehlen des Knickes bei den Kurven 3, 4, 5, 6 entspricht wieder einem allmahlichen Ubergang zur Turbulenzstromung, wie bei den Kapillaren 1-6 im vorigen Abschnitt. Dagegen tritt in der Kurve 1, die einer Windung entspricht, der Knick noch sehr deutlich auf. Der untere Teil 2a besitzt also, wenn wir hier die Reynoldsschen Untersuchungen im Prinzip noch als giiltig annehmen, eine wirbelfreie Stromung, wahrend da- gegen im oberen Teile 2 b Wirbel auftreten. Hauptvereuche. Beide Stromungs- gebiete sind also hier noch scharf getrennt im Gegensatz zu den Kurven 2-6. Der Knick selbst liegt bei einem etwas 632 G. Leehner. kleineren Volumen, d. h. bei einer kleineren mittleren Ge- schwindigkeit. Die Verschiebung ist ganz gering und betragt 5 ccm. Bei den weiteren Kurven laSt sich leider eine Ver- 250 200 100 150 50 Taf. VII. 250 200 100 150 50 Taf. VII. schiebung nicht mehr konstatieren, so daS uber die Abhangig- keit der Verschiebung von der Windungszahl nichts auszusagen ist. Es macht sich daher der EinfluS der Windungen vor allem darin geltend, daS eine Verschiebung der kritischen Ge- schwindigkeit zu kleineren Werten eintritt oder mit anderen Worten, da6 der Eintritt der Turbulenzstromung beschleunigt schiebung nicht mehr konstatieren, so daS uber die Abhangig- keit der Verschiebung von der Windungszahl nichts auszusagen ist. Es macht sich daher der EinfluS der Windungen vor allem darin geltend, daS eine Verschiebung der kritischen Ge- schwindigkeit zu kleineren Werten eintritt oder mit anderen Worten, da6 der Eintritt der Turbulenzstromung beschleunigt Untersuchuny der Turbulenr usw. 633 wird. Ich mochte hier ausdriicklich betonen, daf3 bisher die kritische Geschwindigkeit stets in Reynolds Sinne aufzufassen ist , namlich als derjenigen Grenzgeschwindigkeit, oberhalb welcher in der ganzen Rohre wirbelformige Bewegungen ein- treten, unterhalb welcher aber der Poiseuillesche Stromungs- zustand vorhanden ist. Wenn wir diese Auffassung der kriti- schen Geschwindigkeit auch auf gewundene Rohren ubertragen wollen, bei denen kein unstetiger Ubergang der beiden Be- wegungserscheinungen stattfindet, so konnen wir dies, wenn wir die Rey noldssche Definition im engeren Sinne auffassen. Wir verstehen dann bei gewundenen Kapillaren unter kritischer Geschwindigkeit diejenige, oberhalb welcher in der ganzen Rohre turbulente Encheinungen zutage treten, unterhalb der aber ein allmahlicher Ubergang zwischen Poiseuillescher Stromung und Turbulenz stattfindet. Eine strenge Trennung der beiden Strbmungsgebiete lii6t sich also nicht mehr auf- recht erhalten, was auch schon daraus folgt, daf3 die Neigung der dem Poiseuilleschen Gesetze entsprechenden Kurve immer kleiner wird, also einmal in die konstante Neigung des Turbulenzzweiges ubergehen muB. Diesen Grenzfall habe ich Fig. 10. durch die gestrichelte Kurve 7 (vgl. Taf. VII) dargestellt. Bei ihm treten sofort, also schon bei den niedrigsten Drucken Wirbel auf. Fig. 10. Fig. 10. Fig. 10. durch die gestrichelte Kurve 7 (vgl. Taf. VII) dargestellt. Bei ihm treten sofort, also schon bei den niedrigsten Drucken Wirbel auf. Hauptvereuche. An dieser Stelle mochte ich einige Versuche beschreiben, die sich auf die Einfuhrung von farbigen Flussigkeiten in das Innere der durchstromten Kapillare beziehen, denn man kann an ihnen die Bewegung der Flussigkeit sehr schon beobachten. In eine gerade Rohre mit einem seitlichen Ansatzrohr wurde ein weites in eine sehr feine Spitze auslaufendes Glas- rohr mittels eines Korkes eingesetzt (vgl. Fig. 10). g g g Dieses wurde mit einer Losung von Kaliumpermanganat g g g Dieses wurde mit einer Losung von Kaliumpermanganat Annnlen der Physik. IV. Folge. 42. 41 p 41 p 41 Annnlen der Physik. IV. Folge. 42. 634 G. Lechner. gefullt und das Durchstromungsrohr am einfachsten mit der Wasserleitung verbunden. Bus der feinen Spitze fl06 nun ein sehr diinner Faden in der Mitte der Rohre parallel zur Achse. Erhohte man den Druck, so wurde der Faden immer breiter und endlich nach fjberschreiten des kritischen Druckes erftillte die farbige Flussigkeit die' game Rohre, ein Zeichen dafur, dab bereits Wirbel aufgetreten sind. Den Moment des Ubergangs vom parallelen Stromverlauf zur Turbulenzbewegung konnte man geneu fixieren. Die gleichen Versuche wieder- holte ich mit einer Kapillare von vier Windungen. Ich konnte dabei folgende drei Fillle unterscheiden: gefullt und das Durchstromungsrohr am einfachsten mit der Wasserleitung verbunden. Bus der feinen Spitze fl06 nun ein sehr diinner Faden in der Mitte der Rohre parallel zur Achse. Erhohte man den Druck, so wurde der Faden immer breiter und endlich nach fjberschreiten des kritischen Druckes erftillte die farbige Flussigkeit die' game Rohre, ein Zeichen dafur, dab bereits Wirbel aufgetreten sind. Den Moment des Ubergangs vom parallelen Stromverlauf zur Turbulenzbewegung konnte man geneu fixieren. Die gleichen Versuche wieder- holte ich mit einer Kapillare von vier Windungen. Ich konnte dabei folgende drei Fillle unterscheiden: g 1. Es lie8 sich durch Geschwindigkeitserniedrigung er- reichen, da6 der farbige schmale Faden in dem geradenTeile 1 (vgl. Fig. 10) und der ersten Windung vollstiindig gleichmaBig vorhanden war, wahrend in der zweiten Windung eine Er- weiterung eintrat, die in der dritten zunahm und in der vierten Windung fast. schon das ganze Rohr erfiillte. Bei noch mehr Windungen wiirde dann vollstiindige Turbulenz ein- getreten sein. g 2. Bei weiterer Geschwindigkeitserniedrigung konnte der farbige schmale Faden in dem geraden Teile und in zwei Windungen exakt in der Mitte der Rohre erhalten werden, so da0 also in diesen Teilen noch wirbelfreie Stromung herrschte, wahrend in Windung 3 und 4 allmahliche Verbreiterungen auftreten. 3. Hauptvereuche. SchlieSlich gelang es mir noch, durch fortgesetzte Ver- minderung in drei und schlieblich. in vier Windungen voll- kommene Wirbelfreiheit zu erreichen, was sich daran erkennen lie6, da0 der farbige Stromfaden sich in der Mitte der Rohre durch alle Windungen zog, ohne breiter zu werden. Man sieht also aus diesen Versuchen die aus den Messungen sich ergebendeh Resultate bestiitigt, ja sie gewlhren in mancher Beziehung noch naheren AufschluS. Es ergibt sich namlich gerade aus diesen Versuchen, da6 bei jeder Windungszahl, die unterhalb der dem Grenzfall entsprechenden lie& die wir kritische Windungszahl nennen wollen , ein vollig wirbelfreies Stromungsgebiet existiert. Mit wachsender Windungszahl nimmt dieses Gebiet ab und verschwindet fiir den Qrenzfall ganz, Dieses wirbelfreie Gebiet ist durch eine Grenzgeschwindigkeit 3. SchlieSlich gelang es mir noch, durch fortgesetzte Ver- minderung in drei und schlieblich. in vier Windungen voll- kommene Wirbelfreiheit zu erreichen, was sich daran erkennen lie6, da0 der farbige Stromfaden sich in der Mitte der Rohre durch alle Windungen zog, ohne breiter zu werden. Man sieht also aus diesen Versuchen die aus den Messungen sich ergebendeh Resultate bestiitigt, ja sie gewlhren in mancher Beziehung noch naheren AufschluS. Es ergibt sich namlich gerade aus diesen Versuchen, da6 bei jeder Windungszahl, die unterhalb der dem Grenzfall entsprechenden lie& die wir kritische Windungszahl nennen wollen , ein vollig wirbelfreies Stromungsgebiet existiert. Mit wachsender Windungszahl nimmt dieses Gebiet ab und verschwindet fiir den Qrenzfall ganz, Dieses wirbelfreie Gebiet ist durch eine Grenzgeschwindigkeit Untersuchung der Turbulenr usw. 635 charakterisiert. Beim Uberschreiten derselben tritt nicht plotzliche Wirbelbildung auf, sondern es entstehen zunachst in den dem Aus0uBende nahe gelegenen Windungen Wirbel, die eich mit Erhohung der Geschwindigkeit immer weiter in das Rohreninnere hineinziehen und so einen allmahlichen Uber- gang herstellen. Bei einer bestimmten Geachwindigkeit herrschen Taf. VIII. dann in der ganzen Rohre Wirbel. Und diese ist es, die wir mit der Reynoldsschen kritischen Geschwindigkeit identi- fizieren konnen und die sich nach den oben gemachten Ver- suchen zu kleineren Werten verschiebt. Da, wie aus den Kurventafeln (vgl. Taf. VII) zu ersehen ist, bei mehr als einer Windung kein Knick mehr auftritt, so habe ich es noch mit einer halben Windung von folgender F o r m 1 versucht. In Taf. VIII sind die Ergebnisse zusammengestellt. Did Kurve 2 ent- spricht einer halben Windung, Kurve 3 einer ganzen. Die Lange der Rohre betrug I = 146,6 cm, der Kriimmungshalbmesser 2 p = 5 cm. Hauptvereuche. Da mir am SchluB der Versuche die RBhre zer- sprang, so konnte ich den Radius nicht mehr bestimmen. Die 41 Taf. VIII. dann in der ganzen Rohre Wirbel. Und diese ist es, die wir mit der Reynoldsschen kritischen Geschwindigkeit identi- fizieren konnen und die sich nach den oben gemachten Ver- suchen zu kleineren Werten verschiebt. Da, wie aus den Kurventafeln (vgl. Taf. VII) zu ersehen ist, bei mehr als einer Windung kein Knick mehr auftritt, so habe ich es noch mit einer halben Windung von folgender F o r m 1 versucht. In Taf. VIII sind die Ergebnisse zusammengestellt. Did Kurve 2 ent- spricht einer halben Windung, Kurve 3 einer ganzen. Die Lange der Rohre betrug I = 146,6 cm, der Kriimmungshalbmesser 2 p = 5 cm. Da mir am SchluB der Versuche die RBhre zer- sprang, so konnte ich den Radius nicht mehr bestimmen. Die 41 * 636 G. Lechner. gestrichelte Kurve 1 so11 den Verlauf der geraden Kapillare auf Grund der in den letzten Seiten erlialtenen Resultate un- gefahr andeuten. Man sieht, auch hier bei Kurve 2 und 3 die allerdings kleine Verschiebung der kritischen Geschwindig- keit zu kleineren Werten. Die bisherigen Versuche waren insgesamt mit Kapillaren von sehr kleinem Durchmesser durchgefiihrt. Im Folgenden habe ich Versuche bei sehr grof3em Durchmesser unter An- wendung einer dazu geeigneten Anordnung vorgenommen. Bei diesen Versuchen verwendete ich Leitungswasser. Die Anord- nung selbst sei im folgenden kurz skizziert. Die Rohrleitung L war mit einer Wasserleitung in Ver- Der Hahn HI sowie das verschiebbare bindung gebracht. + Fig. 11. + Fig. 11. Fig. 11. Rohr R dienten zum Regulieren und konstant Halten des Druckes. Die Kapillare K war mit Siegellack an das Mano- meter M gekittet. Der drehbare Hahn S ist ahnlich wie der Schnapphahn in den Hauptversuchen eingerichtet. Von ihm fiihren zwei Leitungen zu den AusfluSgefaBen A, und A, Durch Drehen von S konnte abwechselnd Wasser aus A, oder A, ausflief3en. Die Zeit wahrend des Ausstromens wurde mit einer Stoppuhr gemessen. Die erhaltenen Beobachtungsresul- tate sind in Taf. I X angegeben. Man ersieht hieraus zunachst wieder die Proportionalitat der Abweichungen. Kurve 1 ist die gerade Kapillare, aus ihren Dimensionen 1 = 155,8 cm d = 0,370 cm gezeichnet, Kurve 2 bei einer Windung (Kriim- mungshalbmesser 2 p = 5 cm) und Kurve 3 bei vier Windungen. Ferner ergibt sich hier deutlich die Verschiebung. Hauptvereuche. Bei vier Windungen ist der Knick noch sehr gut erhalten. Es ist daher aus diesen Ergebnissen der Schlut3,zu ziehen, da6 die Verschiebung der kritischen Geschwindigkeit zu kleineren Untersuchung der Turbulenz I S W . 637 Werten um so gro6er ist, je gro6er man den Radius der Ka- pillare wahlt. Dieses Resultat ist auch dadurch gestiitzt, da6 Kapillaren von gro6en Radien den Eintritt der Werten um so gro6er ist, je gro6er man den Radius der Ka- pillare wahlt. Dieses Werten um so gro6er ist, je gro6er man pillare wahlt. Dieses Resultat ist auch dadurch gestiitzt, da6 Kapillaren von gro6en Radien den Eintritt der 0 20 40 Taf. IX. Turbulenzbe w egung mehr begunstigen als enge, da bei ersteren die kritische Geschwin- digkeit sehr klein ist. Eine Krummung wird deshalb bei ihnen zur Turbulenz mehr bei- tragen als bei engen. t age a s be e ge . Was die Abwei- chungen selbst betrifft, so ist zu ihrer Er- klarung anzunehmen, da6 bei gekriimmten Kapillaren nicht mehr der ganze Druck zur fjberwindung der Rei- bungskrafte verwendet wird, sondern nur ein Teil desselben. Der andere Teil dagegen wird zur Uberwindung der infolge der Krum- mung auftretenden Zentrifugalkrafte ver- braucht. Diese An- nahme deckt sich auch mit den Messungs- resultaten, da6 namlich zu ein und demselben Drucke im Falle einer Kriimmung ein kleine- re8 Volumen geh6rt. g mung auftretenden Zentrifugalkrafte ver- braucht. Diese An- nahme deckt sich auch mit den Messungs- resultaten, da6 namlich zu ein und demselben Drucke im Falle einer Kriimmung ein kleine- re8 Volumen geh6rt. 638 G. Lec Da die kritische Geschwin der durch die Kapillare strome war es von Interesse, Versuc zufiihren, deren Dichte von der schieden ist. Zu diesem Zwecke nahm ich Anordnungen fur Quecksilber si ich hierfiir folgende in Fig. 12 I I I I I I I I1 ( 1 I I ; I I I I ; 1 1 I ( I i 1 I strichen in G, zu durchstromen die Differenz zwischen den Mano den Druck, der mit Hilfe von ;.,;, 1; -J 1 1 I I I I I / I I t;. * ' I Fig. 12. 10 638 G L 638 G. Lecher. G. Lecher. Da die kritische Geschwindigkeit auch von der Dichte der durch die Kapillare stromenden Fliissigkeit abhangt, so war es von Interesse, Versuche mit Fliissigkeiten durch- zufiihren, deren Dichte von der des Wassers wesentlich ver- schieden ist. Zu diesem Zwecke nahm ich Quecksilber. Da die friiheren Anordnungen fur Quecksilber sich wenig eigneten, verwandte ich hierfiir folgende in Fig. 12 dargestellte Anordnung. 1; 1y war die Kapil- lare, M, und M, zwei Manometerrohre. Das Glasgefa GI war kali- ~ I briert und konnte eben- I I ' I so wie G, gehoben und I I I 1 1 1 I I I1 I ' 1 1 gesenkt werden. Bei ( 1 I I I ' I I den Versuchen wurde / I die Zeit mit einer ; I I I I ; 1 1 I ( I i 1 ; I Stoppuhr gemessen, I I I / die dae Quecksilber \.---,' brauchte, um ein be- zwischen zwei Teil- strichen in G, zu durchstromen. Der zugehorige Druck war die Differenz zwischen den Manometern Nl und N2. Um in Nl den Druck, der mit Hilfe von G, eingestellt war, moglichst ;.,;, 1; -J 1 1 ,, I I I I I / I I t;. * ' I \ \ I / Fig. 12. stimmtes Volumen ~ I I I ' I I I I 1 1 1 I I I1 I ' 1 1 ( 1 I I I ' I I / I ; I I I I ; 1 1 I ( I i 1 ; I I I I / \.---,' ;.,; 1; -J 1 1 ,, I I I I I / I I t;. * ' I \ \ I / Fig. 12. 10 0 20 Taf. X. Durchmesser: 0,0918 cm Lange: 157,O cm Kriimmungsdurchmesser: 4,s cm. Taf. X. Durchmesser: 0,0918 cm Lange: 157,O cm Kriimmungsdurchmesser: 4,s cm. konstant zu erhalten, nahm ich den Durchmesser von G sehr grob. Ich brauchle daher nur den Enddruck in G, zu be- konstant zu erhalten, nahm ich den Durchmesser von G sehr grob. Ich brauchle daher nur den Enddruck in G, zu be- konstant zu erhalten, nahm ich den Durchmesser von G sehr grob. Ich brauchle daher nur den Enddruck in G, zu be- Untersuchung der Turbulenz usw. 639 obachten. 1’ war ein Thermometer zur Bestimmung der Tempe- ratur des durchstromenden Quecksilbers. Die Resultate sind in den Tafeln X-XIV wiedergegeben. l) E. Gru neisen, Wissenschaftliche Abhandlungen derPhys.Techn. Keichsanstalt 4, Heft 2. G. Lecher. Betrachten wir zunachst Tafel X, so finden wir die friiheren Resultate bestatigt. Kurve 1 entspricht der geraden Rohre, 11 2 ,, zwei Windungen, 71 3 7 7 sechs ,) 1) 5 17 acht 19 Wir haben wieder die lineare Proportionalitat zwischen Abweichung und Windungszahl. Bei Knrve 2 ist der Knick noch vorhanden, wahrend bei den anderen ein allmahlicher Taf. XI. Durchmesser: 0,103 cm, LELnEe: 157,5 cm. Kruinmungedurchmesser: 2,5 cm, Durchmesser: 0,111 em, Lange: 157,6 cm, Krummungsdurchmesser: 2,6 em, Windunpzrhl: 14. Taf. XI. Durchmesser: 0,103 cm, LELnEe: 157,5 cm. Kruinmungedurchmesser: 2,5 cm Taf. XI. Durchmesser: 0,103 cm, LELnEe: 157,5 cm. Kruinmungedurchmesser: 2,5 cm, Durchmesser: 0,111 em, Lange: 157,6 cm, Krummungsdurchmesser: 2,6 em, Windunpzrhl: 14. Durchmesser: 0,111 em, Lange: 157,6 cm, Krummungsdurchmesser: 2,6 em, Windunpzrhl: 14. Durchmesser: 0,111 em, Lange: 157,6 cm, Krummungsdurchmesser: 2,6 em, Windunpzrhl: 14. 640 G Lechnet 640 G. Lechnet. Ubergang stattfindet. Die kritische Geschwindigkeit ist auch hier zu kleineren Werten verschoben. Die Tafeln XI-XIV stellen die in den Vorversuchen benutzten Kapillaren dar. Bei Taf. XIII. Durchmeseer: 0,116 cm, Lange: 157,7 cm, Krummungedurchmeaser: 7,5 cm Winduugszahl: 5. 20 10 0 10 20 30 Taf. XIV. Durchmesser: 0,111 cm, Lange: 157,4 cm, Rrummungsdurchmesser: 7,5 cm, Windungszahl: 5. di i d di d d K ill h d K Taf. XIII. Durchmeseer: 0,116 cm, Lange: 157,7 cm, Krummungedurchmeaser: 7,5 cm Winduugszahl: 5. Taf. XIII. Durchmeseer: 0,116 cm, Lange: 157,7 cm, Krummungedurchmeaser: 7,5 cm Winduugszahl: 5. Durchmeseer: 0,116 cm, Lange: 157,7 cm, Krummungedurchmeaser: 7,5 cm Winduugszahl: 5. 20 10 0 10 20 30 Taf. XIV. Durchmesser: 0,111 cm, Lange: 157,4 cm, Rrummungsdurchmesser: 7,5 cm, Windungszahl: 5. 20 10 0 10 20 30 Taf. XIV. Durchmesser: 0,111 cm, Lange: 157,4 cm, Rrummungsdurchmesser: 7,5 cm, Windungszahl: 5. Durchmesser: 0,111 cm, Lange: 157,4 cm, Rrummungsdurchmesser: 7,5 cm, Windungszahl: 5. diesen sind die der geraden Kapillare entsprechenden Kurven nicht beobachtet, sondern aus bekannten Dimensionen gezeichnet worden. Vergleichen wir die Abweichungen in gleichen Druck- intervallen fur Wasser und Quecksilber, so sehen wir, daB sie diesen sind die der geraden Kapillare entsprechenden Kurven nicht beobachtet, sondern aus bekannten Dimensionen gezeichnet worden. Vergleichen wir die Abweichungen in gleichen Druck- intervallen fur Wasser und Quecksilber, so sehen wir, daB sie 64 1 Untersucitung der Turbulenr usw. fur Wasser geringer sind, eine Tatsache, auf die bereits G r u n - ei s e n l) hinweist. Zueammenfaeeung der Reeultate. Die Versuche haben demnach ergeben, daI3 bei gewundenen Rohren der EinBnI3 der Zentrifugalkraft die Turbulenz ver- griiI3ert und zwar nimmt sie mit der Anzahl der Windungen zu. Das Reynoldssche Kriterium fur die kritische Geschwindig- keit gilt im allgemeinen nicht mehr. Die kritische Ge- schwindigkeit wird zu kleineren Werten verschoben und zwar ist die Verschiebung um so groBer, je gro6er der Kapillaren- radius ist. Von einer verhaltnismaI3ig sehr kleinen Windungs- zahl an existiert iiberhaupt ein strenger Ubergang zur Tur- bulenzbewegung nicht mehr, sondern es findet ein allmahliches Ubergehen statt. Die Beobachtungen mit farbigen Fliissig- keiten zeigen, daf3 die Turbulenz von den letzten Windungen mit zunehmender Geschwindigkeit nach vorne schreitet. Es ist also die Turbulenz auf der ganzen Rohrenlange keine gleich- miiSige, sondern sie nimmt mit Vorriicken der Fliissigkeit zu. g g Wghrend bei einer geraden Reihe mit glatten Wanden zu einer bestimmten Geschwindigkeit immer eine bestimmte Tur- bulenz gehort, die durch den Widerstand, den die Rohre dem Durchgang der Flussigkeit bietet , gemessen wird, ist dieser Widerst,and bei gewundenen Rohren groI3er. 1st Q die in der Seknnde durchgeflossene Fliissigkeitsmenge, P der Druck, so ist P Q=-, W wo der Widerstand CY zunachst nur so lange konstant ist, als das Poiseuillesche Gesetz gilt. Bei Steigerung der Ge- schwindigkeit wird man wieder zu einem konstanten, aber grof3eren Widerstand gefuhrt, wenn man setzt. Hier nimmt Qo mit der Windungszahl ab, wahrend W = d Qld P, sowohl von der Windungsanzahl wie von der Ge- setzt. Hier nimmt Qo mit der Windungszahl ab, wahrend W = d Qld P, sowohl von der Windungsanzahl wie von der Ge- 642 G. Lecher. Untersuchung usw. schwindigkeit unabhangig ist. dann haben wir wieder wie beim Poiseuilleschen Gesetz: SchlieSlich wird Q,, = 0, und Diese Gerade scheint eine Grenzlinie zu sein, unter welche die Volumendrnckkurve nicht zu gehen scheint. Diese Gerade scheint eine Grenzlinie zu sein, unter welche die Volumendrnckkurve nicht zu gehen scheint. Zum Schlusse mochte ich es nicht unterlassen, Hrn. Ge- heimen Hofrat Prof. Dr. W. Wien fUr die Anregung zu dieser Arbeit, sowie fur das stetige Interesse, meinen ergebensten Dank zum Ausdruck zu bringen. (Eingegangen 25. Juni 1913.) (Eingegangen 25. Juni 1913.) (Eingegangen 25. Juni 1913.)
https://openalex.org/W2592012126	https://pure.coventry.ac.uk/ws/files/12088473/Differential_Effects_of_Anxiety_and_Autism.pdf	English	null	Differential effects of anxiety and autism on social scene scanning in males with fragile X syndrome	Journal of neurodevelopmental disorders	2,017	cc-by	7,888	© 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. Differential effects of anxiety and autism on social scene scanning in males with fragile X syndrome Hayley Crawford1,2* , Joanna Moss2,3, Chris Oliver2 and Deborah Riby4 * Correspondence: hayley.crawford@coventry.ac.uk 1Centre for Research in Psychology, Behaviour and Achievement, Coventry University, Coventry CV1 5FB, UK 2Cerebra Centre for Neurodevelopmental Disorders, School of Psychology, University of Birmingham, Edgbaston B15 2TT, UK Full list of author information is available at the end of the article Abstract Background: Existing literature draws links between social attention and socio-behavioural profiles in neurodevelopmental disorders. Fragile X syndrome (FXS) is associated with a known socio-behavioural phenotype of social anxiety and social communication difficulties alongside high social motivation. However, studies investigating social attention in males with FXS are scarce. Using eye tracking, this study investigates social attention and its relationship with both anxiety and autism symptomatology in males with FXS. Methods: We compared dwell times to the background, body, and face regions of naturalistic social scenes in 11 males with FXS (Mage = 26.29) and 11 typically developing (TD) children who were matched on gender and receptive language ability (Mage = 6.28). Using informant-report measures, we then investigated the relationships between social scene scanning and anxiety, and social scene scanning and social communicative impairments. Results: Males with FXS did not differ to TD children on overall dwell time to the background, body, or face regions of the naturalistic social scenes. Whilst males with FXS displayed developmentally ‘typical’ social attention, increased looking at faces was associated with both heightened anxiety and fewer social communication impairments in this group. Conclusions: These results offer novel insights into the mechanisms associated with social attention in FXS and provide evidence to suggest that anxiety and autism symptomatology, which are both heightened in FXS, have differential effects on social attention. Keywords: Eye tracking, Fragile X syndrome, Autism spectrum disorder, Anxiety, Social attention Keywords: Eye tracking, Fragile X syndrome, Autism spectrum disorder, Anxiety, Social attention the protein FMRP. As FXS is an X-linked disorder, males are more severely affected than females. The phenotype associated with FXS encompasses mild to profound intellectual disability alongside physical, cognitive, and behavioural manifestations [2]. Differential effects of anxiety and autism on social scene scanning in males with fragile X syndrome Crawford, H, Moss, J, Oliver, C & Riby, D Original citation: Crawford, H, Moss, J, Oliver, C & Riby, D 2017, 'Differential effects of anxiety and autism on social scene scanning in males with fragile X syndrome' Journal of Neurodevelopmental Disorders, vol 9, 9 http://dx.doi.org/10.1186/s11689-017-9189-6 Original citation: Crawford, H, Moss, J, Oliver, C & Riby, D 2017, 'Differential effects of anxiety and autism on social scene scanning in males with fragile X syndrome' Journal of Neurodevelopmental Disorders, vol 9, 9 http://dx.doi.org/10.1186/s11689-017-9189-6 DOI 10.1186/s11689-017-9189-6 ISSN 1866-1947 ESSN 1866-1955 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. Copyright © and Moral Rights are retained by the author(s) and/ or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This item cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder(s). The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 DOI 10.1186/s11689-017-9189-6 Open Access Background Anxiety is also commonly reported in FXS with over 80% of males meeting criteria for one anxiety disorder and 60% meeting criteria for multiple anxiety disorders. The most common types of anxiety disorder in FXS are specific phobia, selective mutism, and social phobia. Approximately 60% of males with FXS display clinically significant features of social phobia [7]. Despite social communication impairments and social anxiety, individ- uals with FXS are reported to show behaviours suggest- ive of a willingness to interact with others; thus, they appear socially motivated [8–10]. There is a need to utilise ecologically valid social scene stimuli to understand the social attention of males with FXS. Furthermore, given the socio-behavioural profile of the disorder, preliminary insight into the role of anxiety and autistic features is important to understand the poten- tial mechanisms underlying social attention in this group. In typical development, it is known that socially anxious individuals fixate longer on the eye region of faces than those without social anxiety [23]. Anxiety has previously been related to social attention in people with WS, but in a different way, with high levels of anxiety being associated with reduced fixation on faces and eye regions of threaten- ing facial expressions [24]. In FXS, some studies have re- ported that reduced fixation to the eye region of isolated emotionally expressive faces is not associated with social anxiety [20] or autism symptomatology [19, 21], whereas other studies have reported a positive correlation between self-reported social anxiety and time spent looking at the eye region of faces [25]. Studying FXS, a genetic syndrome with heightened risk of autism and anxiety, offers novel insight into the association between these behavioural characteristics and social attention, which may inform understanding of other neurodevelopmental disorders associated with a similar socio-behavioural profile, e.g. ASD and Cornelia de Lange syndrome [26]. pp y Relevant to the features of FXS described above, existing literature within the field of developmental disorders has drawn links between socio-behavioural characteristics and social attention. Research has primarily identified atypic- ally reduced social attention in ASD (behaviourally associ- ated with social withdrawal) and atypically prolonged social attention in Williams syndrome (WS; behaviourally associated with hyper-sociability) [11–14]. Specifically, this research has demonstrated that people with ASD spend less time than typically developing (TD) individuals view- ing people and faces in static pictures of social interaction. Background Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability affecting approximately 1 in 2500 males and 1 in 4000–6000 females [1]. FXS is caused by excessive cytosine-guanine-guanine (CGG) repeats on the Fragile X Mental Retardation 1 (FMR1) gene located on the Xq27.3 site. Individuals with the FXS premutation have 45–200 repeats whereas individ- uals with the full mutation have in excess of 200 repeats. The excessive CGG repeats cause the FMR1 gene to become methylated, resulting in reduced production of FXS is associated with a socio-behavioural phenotype that includes being motivated to interact with others and demonstrating interest in the social world. However, these features co-occur with heightened anxieties and social communication impairments [2, 3]. The social communication impairment associated with FXS is reflected in the heightened prevalence of autism spectrum disorders (ASD). Although prevalence figures often vary across studies, a recent meta-analysis has in- dicated that approximately 30% of males with FXS meet criteria for ASD [4]. This is in comparison to 1% of the * Correspondence: hayley.crawford@coventry.ac.uk 1Centre for Research in Psychology, Behaviour and Achievement, Coventry University, Coventry CV1 5FB, UK 2Cerebra Centre for Neurodevelopmental Disorders, School of Psychology, University of Birmingham, Edgbaston B15 2TT, UK Full list of author information is available at the end of the article Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 Page 2 of 10 Page 2 of 10 general population [5]. However, it is increasingly recog- nised that subtle differences exist between individuals with FXS and those with idiopathic ASD, as those with FXS often display a milder profile of autism symptom- atology. A recent review of existing literature highlights several studies indicating less severe social impairments in individuals with FXS and comorbid ASD compared to individuals with idiopathic ASD, particularly on mea- sures of social responsiveness [6]. scenes reported that a largely female sample of people with FXS spent a ‘typical’ amount of time looking at so- cial information, but that they also looked away quicker than TD participants, indicating active social avoidance [22]. The issue that 12 out of the 14 FXS participants in that study were female is important due to the striking differences in the severity and prevalence of the FXS phenotype between males and females. Therefore, it is problematic to generalise findings from studies using largely female samples to males with FXS who are often more severely affected. Background Attention to social stimuli in this group has also been linked to social behaviour, with reduced social attention being associated with more severe autism symptomatology and consequently more social communication difficulties [15–17]. Much research has focussed on the association between social behaviour and social attention in ASD. However, little is known about the way in which behav- ioural characteristics interact with social attention in males with FXS despite the known social profile associated with this group, and the heightened risk of autism. g y Whilst existing eye-tracking studies in FXS have offered rich information regarding the extent of eye gaze aversion, the current study makes a significant contribution to investigating the influence of anxiety and autism symp- tomatology on social attention in males with FXS using naturalistic social scenes that reflect the complexities of our social world. This study aims to (1) compare and contrast social attention in males with FXS to TD children matched on gender and receptive language ability, (2) in- vestigate the relationship between social attention and anxiety in males with FXS, and (3) investigate the relation- ship between social communication impairment and so- cial attention in males with FXS. Studies that have been conducted in FXS have identi- fied atypical social attention, in the form of reduced looking to the eye region of static isolated faces, com- pared to TD individuals [18–20] and individuals with ASD [20, 21]. However, every one of these studies used isolated face images displaying different emotional ex- pressions. Whilst this offers rich information regarding looking patterns to facial features in FXS, it is known from the literature on both typical development and ASD that such stimuli lack ecological validity as there is no ‘competition’ between social and non-social attention capture (e.g. see discussions by [16]). One study that has investigated social attention to more naturalistic social Participants Participants were 11 males with FXS aged between 14 and 43 years (Mage = 26.29; 9.06). All participants had a Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 Page 3 of 10 Page 3 of 10 confirmed diagnosis from a professional (paediatrician, general practitioner, or clinical geneticist). Participants with FXS were recruited through the Cerebra Centre for Neurodevelopmental Disorders participant database at the University of Birmingham. the camera and displayed natural facial expressions. Spe- cifically, the emotional valence of the actors in the social scenes was mostly neutral, interspersed with a few images where actors were displaying a happy facial expression. The scenery was naturalistic for the activities that actors were engaged in, e.g. classroom, restaurant. Participants also saw five filler photographs of landscapes with no actor, which were interspersed throughout the eye- tracking task so as to avoid a uniform pattern of solely social scenes being displayed. As filler trials contained no social stimuli, eye movements during these trials were not analysed. Stimuli were 640 × 480 pixels. y g Participants with FXS were group-matched to 11 male TD children on receptive language ability (t (20) = −1.208, p = .242) using the raw scores from the British Picture Vocabulary Scale (BPVS; [27]). As previous literature indicates that receptive language is commensurate with nonverbal mental age in adolescents with FXS [28], receptive language was used as a proxy indicator of general intellectual ability. TD children were recruited through the Infant and Child Laboratory participant data- base, also at the University of Birmingham. None of the TD children scored above 15 on the Social Communica- tion Questionnaire (SCQ; [29]), the score suggested by the authors to be indicative of ASD. All of the TD children scored within the normal range on the Spence Child Anx- iety Scale—Parent version (SCAS-P; [30]), defined as the mean + 1 standard deviation, using the national normal data from TD boys aged 6–11 years [31]. The same criter- ion was used to rule out anxiety in children under the age of 6 years in the current study. Table 1 presents the final participant characteristics. Stimuli were presented on a 24-in. widescreen LED monitor at a screen resolution of 1680 × 1050. Participants Partici- pants’ eye movements were recorded using an EyeLink 1000 Tower Mount system, which runs with a spatial accuracy of .5–1 visual angle (°), a spatial resolution of .01°, and a temporal resolution of 500 Hz. The right eye of each participant was tracked. The eye-tracking camera was linked to a host PC separate to the one displaying the stimuli. EyeLink software (SR research, Ontario, Canada) was used to control the camera and collect data. Measures The participants' primary caregivers completed the SCQ [29] and the SCAS-P [30] to measure social communica- tion impairments and anxiety, respectively, and for the purposes of investigating associations between these behavioural characteristics and social attention in the present study. The SCAS-P assesses the following six domains of anxiety: physical injury fears, obsessive- compulsive disorder, separation anxiety, social phobia, panic/agoraphobia, and generalised anxiety, and has been shown to differentiate those with and without an anxiety disorder. Internal consistencies of the total scale and subscales range from .83 to .92 in an anxiety- disordered group and .81 to .90 in typical controls. The SCAS-P total score correlates significantly with the Child Behavior Checklist [32] internalising subscale, indicating convergent validity [31]. Caregivers completed these measures either whilst their child was participating in the study or at home, returning it to the researchers All participants had normal or corrected to normal vision. All participants aged 16 years and over provided informed written consent, and parents of children aged under 16 provided written consent before taking part in the study, in line with the ethical approval granted from the Science, Technology, Engineering and Mathematics Ethical Review Committee at the University of Birmingham. Procedure Participants were tested individually at the University of Birmingham in a dimly lit room with windows blacked out to avoid luminance changes. Participants were seated approximately .6 m from the screen with their chin rest- ing on a chinrest and their forehead against a headrest. The chinrest and desk height were adjusted so that eye gaze was central to the display screen. A 5-point calibra- tion was performed prior to the experiment during which participants followed the location of an animated blue dolphin positioned at the edges of the display area. The calibration procedure was repeated until successful, and all participants included in the analysis achieved a full 5-point calibration. Following calibration, the parti- cipants were told that they would view a series of pictures and that they could look wherever they wished whilst these were displayed. Each image was then presented for 5 s. Between each trial, a fixation cross appeared at the centre of the screen for 1 s. ( g ) A 3 (AOI: background, body, face) × 2 (group: FXS, TD) ANOVA was conducted, which revealed a signifi- cant main effect of AOI (F (2, 40) = 38.153, p < .001, n2 = .656) but no significant main effect of group (F (1, 20) = .009, p = .923, n2 < .001), and no significant inter- action (F (2, 40) = 1.066, p = .354, n2 = .051). Bonferroni post hoc tests indicated that the main effect of AOI was driven by longer dwell time on the background than the body and the face regions of the actors in the scenes (both p < .001). Dwell times on the face and body region of actors were statistically comparable (p = .081). However, Wilcoxon signed rank tests, which were conducted be- cause body AOI data were not normally distributed, re- vealed longer dwell time on the face compared to the body region of actors (Z = −2.029, p = .042). y g p Correlations were conducted to assess the association between dwell time on each AOI and social communica- tion difficulties, as measured by the SCQ, and social phobia and total anxiety scores, as measured by the SCAS-P for each participant group. Table 2 shows the descriptive statistics for these measures by group. Stimuli and apparatus The stimuli used were identical to those used by Riby and Hancock [11]. Stimuli consisted of 20 colour photo- graphs of naturalistic social scenes including human actors engaged in natural activities. Example scenes included a bride and groom on their wedding day, a woman on the phone, a group of friends talking to one another, and a teacher in a classroom. Actors in the photographs were not directing their attention towards Table 1 Participant characteristics and alpha level for comparison between FXS and TD participants FXS (n = 11) TD (n =11) t df p Chronological age (years) Mean (SD) 26.29 (9.06) 6.28 (1.31) −7.256 20 <.001 Range 14.12–43.01 4.60–8.94 Receptive language ability (raw score) Mean (SD) 87.00 (27.21) 74.18 (22.32) −1.208 20 .241 Range 87–135 47–114 Gender (% male) 100 100 Comparison between participants on chronological age, receptive language ability as measured by the British Picture Vocabulary Scale, and gender Table 1 Participant characteristics and alpha level for comparison between FXS and TD participants FXS (n = 11) TD (n =11) t Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 Page 4 of 10 used where data are normally distributed. The alpha level for significance was .05. on completion. All participants lived at home with the caregiver completing the questionnaire measures. The Autism Diagnostic Observation Schedule (ADOS; [33]) was administered to all participants with FXS for diag- nostic purposes (module 2: n = 2; module 3: n = 5; mod- ule 4: n = 4). The BPVS [27] was administered to all participants to assess receptive language ability. Procedure A significant negative correlation between SCQ score and dwell time to the background was revealed for the TD group (rs (7) = −.792, p = .011), indicating that those indi- viduals with fewer social communication difficulties spent more time looking at the background. No other significant correlations were revealed for the TD partici- pant group (all p > .05; Table 3). For participants with FXS, moderate-strong positive correlations were revealed between dwell time on the face AOI and social phobia (rp (8) = .687, p = .028; Fig. 2), and between dwell time on the face AOI and total anxiety score (rp (8) = .742, p = .014; Fig. 3). A significant negative correlation was revealed be- tween dwell time on the face AOI and SCQ score (rp (7) = −.720, p = .029; Fig. 4). This did not remain significant after controlling for receptive language ability (rp (5) = −.704, p = .077). Taken together, this indicates that those FXS participants with higher anxiety scores, and fewer social communication difficulties, exhibited lon- ger dwell times on faces. Results There was no difference in the overall amount of time participants spent viewing stimuli, indicating comparable task engagement across the groups (FXS mean per image: 4202.46 ms; TD mean per image: 4237.88 ms; t (20) = .148, p = .884). The remaining analyses concern dwell time in milliseconds for each AOI (see Fig. 1). Data analysis Areas of interest (AOI) were designated to the face, body, and background using the Data Viewer programme (SR Research). Face and body AOI were created using the FreeHand Interest Area Shape to select the outline of each actor’s face and body. The background AOI was created using the Rectangular Interest Area Shape, to cover the entire image, and then subtracting fixation data from the face and body AOI prior to analysis. Data are presented as the total time, in milliseconds, that fixations were within each AOI. A trial was deemed invalid, and therefore ex- cluded, if a participant did not look at the picture pre- sented for any of the trial time. If any participant produced more than 40% invalid trials, their data were excluded from analyses. In the current study, one par- ticipant produced one invalid trial only. Therefore, no participants were excluded due to insufficient data. All data were subjected to the Shapiro-Wilk test for nor- mality. Where data were not normally distributed, non- parametric tests were used for statistical analyses. For the between-group comparisons, where results from non-parametric tests did not differ from results from the equivalent parametric tests, the results from the parametric tests are reported. For within-group correla- tions, Spearman’s correlations are used where data are not normally distributed and Pearson’s correlations are As participant groups were not matched on chrono- logical age, correlations were conducted to assess the relationship between chronological age and dwell time, especially due to the large age range of the FXS group. Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 Page 5 of 10 Fig. 1 Dwell time on AOIs; dwell time in milliseconds on background, body, and face AOI for the FXS and TD participant groups, when overall engagement with the stimuli did not differ across groups Fig. 1 Dwell time on AOIs; dwell time in milliseconds on background, body, and face AOI for the FXS and TD participant groups, when overall engagement with the stimuli did not differ across groups These revealed no significant association between chrono- logical age and dwell time on any AOI for either partici- pant group (all p > .05). Although participant groups were matched on receptive language ability, correlations were conducted to assess the relationship between receptive language and dwell time in the event that our group- matching comparison was underpowered. Data analysis These re- vealed no significant association between receptive language and dwell time on any AOI for either partici- pant group (all p > .05). Discussion In the present study, we examined and compared visual attention to naturalistic social scenes in males with FXS versus TD individuals. In addition, we investigated the relationship between social attention, anxiety, and social communication difficulties. The results demonstrated statistically comparable dwell time on background, body, and face regions of the social scenes across the two participant groups. The results also demonstrated an association between increased looking at faces with Table 2 Descriptive statistics and alpha level for the ADOS, SCQ, and SCAS-P measures Measure FXS TD t df p ADOS Mean raw total score (SD) 8.64 (5.12) NA Range 2–22 % meeting cut-off for ASD 72.73 % meeting cut-off for autism 18.18 Social Communication Questionnairea Mean raw total score (SD) 17.57 (6.27) 2.89 (2.37) −6.569 16 <.001 Range 6–27 0–6 % meeting cut-off for ASD 77.7 0 % meeting cut-off for autism 22.22 0 Spence Child Anxiety Scaleb Mean raw Social Phobia score (SD)c 4.33 (4.53) 2.63 (2.26) −.967 16 .348 Range 0–14.4 0–6 Mean raw total score (SD)d 19.54 (16.95) 9.38 (5.32) −1.625 16 .124 Range 1–49 1–20 aSCQ was not completed for two TD participants and two FXS participants bSCAS-P was not completed for three TD participants and one FXS participant cThe maximum Social Phobia score on the SCAS-P is 18. Normative data obtained from Nauta et al. [31] indicate a mean score of 7.3 for anxiety-disordered and a mean score of 4.3 for typically developing boys aged 6–11 years dThe maximum total score on the SCAS-P is 114. Normative data obtained from Nauta et al. [31] indicate a mean total score of 31.4 for anxiety-disordered and 16.0 for typically developing boys aged 6–11 years Crawford et al. Discussion Journal of Neurodevelopmental Disorders (2017) 9:9 Page 6 of 10 Table 3 Correlations between behavioural characteristics and social attention, and between participant characteristics and social attention Table 3 Correlations between behavioural characteristics and social attention, and between participant characteristics and social attention Fragile X syndrome Typically developing Face rp (p) Body rp (p) Background rp (p) Face rp (p) Body rs (p) Background rs (p) Social phobia .687 (.028) −.311 (.981) −.161 (.657) −.059 (.890) −.024 (.955) −.539 (.168) Total anxiety score .742 (.014) −.153 (.673) −.250 (.486) −.265 (.525) .120 (.776) −.663 (.073) Total SCQ score −.720 (.029) .077 (.845) .099 (.800) −.660 (.053) .017 (.965) −.792 (.011) Chronological age .593 (.055) −.105 (.758) .165 (.627) .166 (.627) −.082 (.811) .191 (.574) Receptive language .383 (.246) .073 (.831) .422 (.196) −.178 (.601) .178 (.601) .483 (.132) Correlation matrix for correlations between dwell time on face, body, and background AOI with (1) social phobia, as measured by the SCAS-P, (2) total anxiety score on the SCAS-P, (3) social communication impairment, as measured by the SCQ, (4) chronological age, and (5) receptive language raw score, as measured by the BPVS Table 3 Correlations between behavioural characteristics and social attention, and between participant characteristics and between behavioural characteristics and social attention, and between participant characteristics and Correlation matrix for correlations between dwell time on face, body, and background AOI with (1) social phobia, as measured by the SCAS-P, (2) total anxiety score on the SCAS-P, (3) social communication impairment, as measured by the SCQ, (4) chronological age, and (5) receptive language raw score, as measured by the BPVS for the results presented here, documenting that these individuals do not show reduced social attention in the same way as those with ASD. Existing literature suggests that individuals with FXS demonstrate less severe im- pairments in social responsiveness compared to individ- uals with ASD, even when matched on overall autism severity [6, 34]. These different profiles go some way to explaining why reduced social attention may be expected in individuals with ASD but not in those with FXS. increased anxiety and fewer social communication diffi- culties in individuals with FXS. Together, these results suggest that whilst social attention to naturalistic social scenes may be developmentally ‘typical’ in males with FXS, anxiety and autism symptomatology are differen- tially related to social attention in this population. Discussion Existing studies that have indicated atypical social attention in males with FXS have focussed on attention to the eye region of static faces. However, the current study revealed that social attention to naturalistic social scenes appears developmentally ‘typical’ in males with FXS. A number of important advances have indicated reduced social attention in individuals with ASD, which is associated with social withdrawal [11–14]. The milder profile of social communication difficulties, and subtle but important differences in the social impairment reported in individuals with FXS [2, 3, 6], may account Although there were no significant differences be- tween the FXS and TD groups in relation to overall looking time, increased looking to faces was correlated with fewer social communication difficulties in individ- uals with FXS. This is a finding that is often reported in the ASD literature [15–17], and one that suggests autism symptomatology may play a role in the viewing of natur- alistic social scenes. Interestingly, in our previous work Fig. 2 Relationship between face AOI and social anxiety; a scatterplot depicting the relationship between dwell time on the face AOI in milliseconds, and the SCAS-P social phobia score for participants with FXS. The analyses indicate a significant positive correlation (rp (8) = .687, p = .028) Fig. 2 Relationship between face AOI and social anxiety; a scatterplot depicting the relationship between dwell time on the face AOI in milliseconds, and the SCAS-P social phobia score for participants with FXS. The analyses indicate a significant positive correlation (rp (8) = .687, p = .028) Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 Page 7 of 10 Fig. 3 Relationship between face AOI and anxiety; a scatterplot depicting the relationship between dwell time on the face AOI in milliseconds, and the SCAS-P total score for participants with FXS. The analyses indicate a significant positive correlation (rp (8) = .742, p = .014) Fig. 3 Relationship between face AOI and anxiety; a scatterplot depicting the relationship between dwell time on the face AOI in milliseconds, and the SCAS-P total score for participants with FXS. The analyses indicate a significant positive correlation (rp (8) = .742, p = .014) region of faces than those without social anxiety [23]. This potential explanation is supported by our previous eye-tracking study, which revealed a positive relationship between social dwell time on videos of actors approaching the viewer, and anxiety, in males with FXS [35]. Discussion The results of the current study are also interesting in light of existing behavioural observation research that highlighted a pattern of results in which more eye contact was asso- ciated with increased cortisol reactivity, a physiological indicator of stress, in individuals with FXS [36]. It is important to note that although the mean anxiety scores for participants with FXS did not differ from normative data from TD children, within-syndrome variability was large. Participants with FXS were therefore more likely to achieve scores on the SCAS-P indicative of more severe anxiety than children with an anxiety disorder (see [31] for normative data). directly comparing individuals with FXS and ASD, we reported that atypical eye gaze in FXS was not a product of autistic symptomatology [21]. Together, these results suggest that social attention to naturalistic scenes ap- pears developmentally typical but may be influenced by autism symptomatology, whereas eye gaze aversion is a FXS-specific impairment that is unlikely to be a product of autism symptomatology in the same way. The current study reported a relationship between heightened looking at faces and anxiety. A potential mechanism underlying this explanation is that individ- uals experiencing anxiety, and social anxiety in particu- lar, may view faces as a more threatening aspect of a social scene. Therefore, heightened looking to threaten- ing stimuli may reflect hyper-vigilance for threatening stimuli, supporting previous literature indicating that socially anxious TD individuals fixate longer on the eye Fig. 4 Relationship between face AOI and autism symptomatology; a scatterplot depicting the relationship between dwell time on the face AOI in milliseconds and the SCQ total score for participants with FXS. The analyses indicate a significant negative correlation (rp (7) = −.720, p = .029) Fig. 4 Relationship between face AOI and autism symptomatology; a scatterplot depicting the relationship between dwell time on the face AOI in milliseconds and the SCQ total score for participants with FXS. The analyses indicate a significant negative correlation (rp (7) = −.720, p = .029) Page 8 of 10 Page 8 of 10 Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 The differential relationships reported here, between social attention and both anxiety and autism symptom- atology, are particularly interesting when existing litera- ture on WS is considered. Discussion Less time spent looking at the eye region of faces has been related to higher levels of autism symptomatology in individuals with WS [37], a similar relationship to that reported in the current study where less looking at faces was associated with higher levels of autism symptomatology. Additionally, increased levels of generalised anxiety have been associated with reduced fixation on faces and eyes for individuals with WS [24], which is the opposite pattern of results to that reported in the current FXS sample where increased levels of anxiety were associated with increased dwell time on faces. One possible explanation for these cross-syndrome differences in the relationship between social attention and anxiety may be related to the different profiles of anxiety associated with these two genetic syndromes. Al- though both FXS and WS are associated with high levels of specific phobia, FXS is also typically associated with so- cial anxiety [7] whilst WS is associated with generalised anxiety disorder [38]. Such cross-syndrome insights allow us to advance our understanding of syndrome-specific mechanisms that might underlie social attention patterns. more flexibly at mouths (especially when talking) and hands (especially when picking up an object) [39]. The development of social attention across childhood and adolescence has focussed on specific skills such as facial expression recognition, which seems to improve with age [40, 41]. Less is known about the effect of age and social experience on social attention in a passive viewing task. It is important to note that the sample size and age range in the current study is similar to that of other eye- tracking studies investigating social attention in FXS [18–20, 42]. However, further research in this area is required to clarify the nature of social attention to na- turalistic social stimuli in males with FXS, and to di- sentangle the effects of developmental level and other behavioural characteristics, such as social communica- tion impairments and anxiety, on social attention. Furthermore, although IQ measures were not adminis- tered for the present study due to methodological im- practicality of administering multiple different IQ tests to account for the wide range of ages and abilities of participants, the two participant groups were matched on receptive language. Receptive language has been reported to be commensurate with nonverbal mental age in adolescents with FXS [28]. It is possible that the statistical test to confirm that groups were matched was underpowered. Discussion To that end, receptive language ability was taken into account with our statistical tests, and cor- relations between receptive language and social attention were not significant. Finally, although genetic reports were not available for the current study, future research could investigate the relationship between genetic fac- tors and social attention. Interestingly, our previous work has demonstrated a relationship between genetic variation and visual scanning of emotional faces [43]. Overall looking time indicated good levels of task en- gagement by both groups, highlighting the opportunities afforded by using eye tracking to investigate the mechanisms subserving clinically relevant behaviours in males with FXS. It is essential to apply caution when interpreting the results of the present study due to the small sample sizes. However, moderate to strong correlations between social attention, anxiety, and social communication im- pairments were revealed even with these small samples, highlighting the potential utility of further investigations in this area. The scatterplots (Figs. 2 and 3) indicate further that the significant correlations are unlikely to be driven by outliers. Whilst the between-group com- parisons may have been statistically underpowered, the alpha levels are well above the significance cut-off (group × AOI interaction: p = .354; between-group comparisons: p = .923). Therefore, it seems unlikely that these results would differ with additional participants. p p In addition, the wide age range of the FXS group should be considered when interpreting the results due to the possibility of age-related differences in social attention and behavioural characteristics. Group match- ing on chronological versus mental age is a common issue in intellectual disability research, and we, therefore, suggest our results indicate developmentally ‘typical’ social attention in FXS. The extent to which social atten- tion in the FXS group would compare to individuals of the same chronological age is beyond the scope of this study. However, correlations to investigate the relation- ship between chronological age and social attention were not significant. Existing literature has reported inte- resting differences in social attention as a function of chronological age, with children aged 3 months looking more at eyes, and older children aged 30 months looking Funding The research reported here was supported by a grant from the Economic and Social Research Council (Grant Number: ES/I901825/1) and by Cerebra. 11. Riby DM, Hancock PJB. Viewing it differently: social scene perception in Williams syndrome and autism. Neuropsychologia. 2008;46:2855–60. 12. Riby DM, Hancock PJB. Looking at movies and cartoons: eye-tracking evidence from Williams syndrome and autism. J Intellect Disabil Res. 2009;53:169–81. Competing interests Competing interests The authors declare that they have no competing interests. 18. Dalton KM, Holsen L, Abbeduto L, Davidson RJ. Brain function and gaze fixation during facial-emotion processing in fragile X and autism. Autism Res. 2008;1:231–9. Availability of data and materials The data that support the findings of this study are not available due to them containing information that could compromise research participant consent. 13. Riby DM, Doherty-Sneddon G, Bruce V. Exploring face perception in disorders of development: evidence from Williams syndrome and autism. J Neuropsychol. 2008;2:47–64. Abbreviations children in South Thames: The Special Needs and Autism Project (SNAP). Lancet. 2006;368:210–5. children in South Thames: The Special Needs and Autism Project (SNAP). Lancet. 2006;368:210–5. ADOS: Autism Diagnostic Observation Schedule; ANOVA: Analysis of variance; AOI: Area of interest; ASD: Autism spectrum disorder; BPVS: British Picture Vocabulary Scale; CGG: Cytosine-guanine-guanine; FMR1: Fragile X Mental Retardation 1 gene; FMRP: Fragile X Mental Retardation Protein; FXS: Fragile X syndrome; SCAS-P: Spence Child Anxiety Questionnaire—Parent Version; SCQ: Social Communication Questionnaire; TD: Typically developing; WS: Williams syndrome 6. Abbeduto L, McDuffie A, Thurman AJ. The fragile X syndrome-autism comorbidity: what do we really know? Front Genet. 2014;5:355. 7. Cordeiro L, Ballinger E, Hagerman R, Hessl D. Clinical assessment of DSM-IV anxiety disorders in fragile X syndrome: prevalence and characterization. J Neurodev Disord. 2011;3:57–67. 8. Roberts JE, Weisenfeld LAH, Hatton DD, Heath M, Kaufmann WE. Social approach and autistic behavior in children with fragile X syndrome. J Autism Dev Disord. 2007;37:1748–60. Conclusions The present study documents differential effects of anx- iety and autism on social attention in males with FXS. To our knowledge, this is the first study to investigate visual attention to naturalistic social scenes in a sample of males with FXS. This offers insights into the potential mechanisms subserving social attention in this popula- tion and how this might differ to other genetically de- fined neurodevelopmental disorders. The research paves the way for future investigations of the relationship between clinically relevant, socio-behavioural pheno- types, and social attention, in theories of social attention in neurodevelopmental disorders. Page 9 of 10 Page 9 of 10 Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 Authors’ contributions 14. Riby DM, Hancock PJB. Do faces capture the attention of individuals with Williams syndrome or autism? Evidence from tracking eye movements. J Autism Dev Disord. 2009;39:421–31. HC participated in the study design and coordination, performed the statistical analyses, participated in the interpretation of the data, and drafted the manuscript. JM participated in the study coordination, interpretation of the data, and helped to draft the manuscript. CO participated in the study coordination, interpretation of the data, and helped to draft the manuscript. DR conceived of the study, participated in its design and coordination, participated in the interpretation of the data, and helped to draft the manuscript. All authors read and approved the final manuscript. 15. Klin A, Jones W, Schultz R, Volkmar F, Cohen D. Visual fixation patterns during viewing of naturalistic social situations as predictors of social competence in individuals with autism. Arch Gen Psychiatry. 2002;59:809–16. 16. Speer LL, Cook AE, McMahon WM, Clark E. Face processing in children with autism effects of stimulus contents and type. Autism. 2007;11:265–77. 17. Kliemann D, Dziobeck I, Hatri A, Steimke R, Heekeren HR. Atypical reflexive gaze patterns on emotional faces in autism spectrum disorders. J Neurosci. 2010;30:12281–7. Acknowledgements 9. Kau ASM, Tierney E, Bukelis I, Stump MH, Kates WR, Trescher WH, Kaufmann WE. Social behavior profile in young males with fragile X syndrome: characteristics and specificity. Am J Med Genet A. 2004;126:9–17. g The research reported here was supported by a grant from the Economic and Social Research Council (Grant Number: ES/I901825/1) and by Cerebra. The authors would like to thank all of the participants and their families and Professor Gaia Scerif for her contribution to the interpretation of the data. 10. Kaufmann WE, Cortell R, Kau ASM, Bukelis I, Tierney E, Gray RM, Cox C, Capone GT, Stanard P. Autism spectrum disorder in fragile X syndrome: communication, social interaction, and specific behaviors. Am J Med Genet A. 2004;129:225–34. Ethics approval and consent to participate 20. Holsen LM, Dalton KM, Johnstone T, Davidson RJ. Prefrontal social cognition network dysfunction underlying face encoding and social anxiety in fragile X syndrome. Neuroimage. 2008;43:592–604. Ethical approval for the current study was obtained from the School of Psychology Ethical Review Board at the University of Birmingham. y gy y g Participants aged 16 and above provided written consent to participate. y gy y g Participants aged 16 and above provided written consent to participate. P f hild d b l 6 id d i b h lf 21. Crawford H, Moss J, Anderson GM, Oliver C, McCleery JP. Implicit discrimination of basic facial expressions of positive/negative emotion in fragile X syndrome and autism spectrum disorder. Am J Intellect Dev Disabil. 2015;120:328–45. Parents of children aged below 16 years provided written consent on behal of their children. Parents of children aged below 16 years provided written consent on behalf of their children. Received: 1 September 2016 Accepted: 10 February 2017 25. Shaw TA, Porter MA. Emotion recognition and visual-scan paths in fragile X syndrome. J Autism Dev Disord. 2013;43:1119–39. 25. Shaw TA, Porter MA. Emotion recognition and visual-scan paths in fragile X syndrome. J Autism Dev Disord. 2013;43:1119–39. Consent for publication Not applicable. 19. Farzin F, Rivera SM, Hessl D. Brief report: visual processing of faces in individuals with fragile X syndrome: an eye tracking study. J Autism Dev Disord. 2009;39:946–52. Author details 1 22. Williams TA, Porter MA, Langdon R. Viewing social scenes: a visual scan-path study comparing fragile X syndrome and Williams syndrome. J Autism Dev Disord. 2013;43:1880–94. 22. Williams TA, Porter MA, Langdon R. Viewing social scenes: a visual scan-path study comparing fragile X syndrome and Williams syndrome. 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Autism Diagnostic Observation Schedule: Manual. Los Angeles: Western Psychological Services; 1999. 34. McDuffie A, Thurman AJ, Hagerman RJ, Abbeduto L. Symptoms of autism in males with fragile X syndrome: a comparison to nonsyndromic ASD using current ADI-R scores. J Autism Dev Disord. 2014;45:1925–37. 35. Crawford H, Moss J, Oliver C, Elliott N, Anderson GM, McCleery JP. Visual preference for social stimuli in individuals with autism or neurodevelopmental disorders: an eye-tracking study. Mol Autism. 2016;7:24. 36. Crawford et al. Journal of Neurodevelopmental Disorders (2017) 9:9 References Hessl D, Glaser B, Dyer-Friedman J, Reiss AL. Social behavior and cortisol reactivity in children with fragile X syndrome. J Child Psychol Psychiatry. 2006;47:602–10. 37. Hanley M, Riby D, Caswell S, Rooney S, Back E. Looking and thinking: how individuals with Williams syndrome make judgements about mental states. Res Dev Disabil. 2013;34:4466–76. 38. Dykens EM. 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W3177383838.txt	https://jneuroinflammation.biomedcentral.com/counter/pdf/10.1186/s12974-021-02178-z	en		Divanillyl sulfone suppresses NLRP3 inflammasome activation via inducing mitophagy to ameliorate chronic neuropathic pain in mice	Journal of neuroinflammation	2,021	cc-by	9,328	Shao et al. Journal of Neuroinflammation (2021) 18:142 https://doi.org/10.1186/s12974-021-02178-z RESEARCH Open Access Divanillyl sulfone suppresses NLRP3 inflammasome activation via inducing mitophagy to ameliorate chronic neuropathic pain in mice Shuai Shao†, Cheng-Bo Xu†, Cheng-Juan Chen, Gao-Na Shi, Qing-Lan Guo, Yu Zhou, Ya-Zi Wei, Lei Wu, Jian-Gong Shi* and Tian-Tai Zhang* Abstract Background: Chronic neuropathic pain is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Divanillyl sulfone (DS), a novel structural derivative of 4,4′-dihydroxydibenzyl sulfoxide from a traditional Chinese medicine Gastrodia elata with anti-nociceptive effects, significantly alleviated neuropathic pain following intrathecal injection. Here, we aimed to investigate the underlying mechanisms of DS against neuropathic pain. Methods: A chronic constrictive injury (CCI) mouse model of neuropathic pain induced by sciatic nerve ligation was performed to evaluate the effect of DS by measuring the limb withdrawal using Von Frey filament test. Immunofluorescence staining was used to assess the cell localizations and expressions of Iba-1, ASC, NLRP3, and ROS, the formation of autolysosome. The levels of NLRP3-related proteins (caspase-1, NLRP3, and IL-1β), mitophagy-related proteins (LC3, Beclin-1, and p62), and apoptosis-related proteins (Bcl-XL and Bax) were detected by Western blotting. The apoptosis of BV-2 cell and caspase activity were evaluated by flow cytometry. Results: DS significantly alleviated the neuropathic pain by increasing the mechanical withdrawal threshold and inhibiting the activation of NLRP3 in CCI-induced model mice. Our findings indicated that DS promoted the mitophagy by increasing the LC3II and Beclin 1 and decreasing the levels of p62 protein in BV-2 cell. This is accompanied by the inhibition of NLRP3 activation, which was shown as inhibited the expression of NLRP3 in lysates as well as the secretion of mature caspase-1 p10 and IL-1β p17 in supernatants in cultured BV-2 microglia. In addition, DS could promote mitophagy-induced improvement of dysfunctional mitochondria by clearing intracellular ROS and restoring mitochondrial membrane potential. Conclusion: Together, our findings demonstrated that DS ameliorate chronic neuropathic pain in mice by suppressing NLRP3 inflammasome activation induced by mitophagy in microglia. DS may be a promising therapeutic agent for chronic neuropathic pain. Keywords: Chronic neuropathic pain, Microglia, Mitophagy, NLRP3 inflammasome, Divanillyl sulfone * Correspondence: shijg@imm.ac.cn; ttzhang@imm.ac.cn † Shuai Shao and Cheng-Bo Xu contributed equally to this work. State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shao et al. Journal of Neuroinflammation (2021) 18:142 Background Neuralgia is a type of chronic pain that manifests as spontaneous pain, hyperalgesia, and allodynia [1, 2]. It is caused by injury or disease in the somatosensory nervous system that includes the central nerves, spinal cord, posterior root of the spinal cord, and peripheral nerves. Pre-clinical studies show that the basis of neuropathic pain is the plasticity of nerve cells [3, 4]. In addition, some non-neuronal cells of the central nervous system (CNS), especially microglia, have also been implicated in triggering neuropathic pain [5–8]. Microglia is a kind of primary innate immune cells in the CNS, and is activated by various pathological stimuli. The activated microglia interacts with the astroglia or neurons to induce neuroinflammation and facilitate transmission of pain signals [9–11]. Chronic neuropathic pain is characterized by infiltration of immune cells into the dorsal root ganglia (DRG), and the activation of microglia in spinal cord and brain, eventually leading to a neuroinflammatory response [12]. Pro-inflammatory factor of interleukin-1β (IL-1β) plays an important role in the microglial inflammatory signaling mediated neuropathic pain [13]. Multiple mechanisms participate in the central neuronal excitation mediated by microglial inflammation in peripheral nerve injury (PNI)-induced neuropathic pain. In microglia, the activation of toll-like receptor 2 (TLR2) and/or TLR4 can promote nuclear factor-κB (NF-κB) signal to induce IL-1β transcription [14], triggering receptor expressed on myeloid cells 2 (TREM2) and via transcription factors interferon regulatory factor 1/5/8 (IRF1/5/8) [15]. In addition, P2X purinoceptor 7 (P2X7), P2Y purinoceptor 12 (P2Y12), and CX3C-chemokine receptor 1 (CX3CR1) on microglial cells also lead to IL-1β secretion via p38 MAPKs as well [16]. The important thing is that nucleotide-binding oligomerization domain (NOD), leucine-rich repeat, and pyrin domains-containing protein (NLRP)-type inflammasomes promote pro-IL-1β processing and mature IL1β secretion. Of NLR family, NLR family pyrin domaincontaining 3 (NLRP3) is the most extensively studied and well-characterized inflammasome sensor molecule. Meanwhile, NLRP3 inflammasome can also be activated by TLRs receptor and P2X7. IL-1β acts as mediators between microglia and neurons and assumes roles as neuromodulator when it acts on spinal dorsal horn (SDH) neurons to increase the strength of synaptic connectivity and excitatory synaptic transmission in the process of neuropathic pain [17, 18]. Thereby, the NLRP3 inflammasome is the most recognized contributor and plays an irreplaceable role to the transmission of pain signals [19, 20]. NLRP3 inflammasome is a multi-protein complex consisting of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) Page 2 of 18 and danger-associated molecular patterns (DAMPs) during the course of natural immune response, which then activates caspase-1 and promotes the maturation and secretion of pro-inflammatory cytokines like IL-1β and interleukin18 (IL-18) [16, 21]. The NLRP3 inflammasome/IL-1 pathway shows outstanding advantages in the process of neuralgia signal transduction. The IL-1β secreted from the microglia binds to the IL-1 receptor (IL-1R) expressed on SDH neurons, which promotes N-methyl-D-aspartate receptor (NMDAR) phosphorylation and reverses γaminobutyrate (GABA) and/or glycine-mediated synaptic inhibition, thereby enhancing transmission of excitatory signals [11, 22, 23]. IL-1R has two isoforms of IL-1R1 and IL1R2. In contrast to IL-1R1, IL-1R2 is a decoy receptor and negative regulator of the IL-1 pathway, just like IL-1R antagonist (IL-1Ra). IL-1Ra, one of the members of IL-1 family acting as a natural IL-1 inhibitor, could bind to both IL1R1 and IL-1R2 to prevent uncontrolled activation of IL-1 receptors by competing IL-1β or IL-1α for binding [24]. Although IL-1Ra or IL-1β neutralizing antibodies are promising analgesics [25–27], complete blockade of IL-1β/IL-1R1 will have an immunosuppressive effect that may increase the risk of infections [17]. Therefore, selective inhibition of IL-1β secretion by activated NLRP3 inflammasome is a better strategy to alleviate neuropathic pain, since the noncanonical pathway of NLRP3 activation and other inflammasomes can still ensure IL-1β secretion for normal physiological functions [28]. To this end, direct inhibitors of NLRP3 inflammasome or activation pathway blockers are increasingly gaining attention for their potential analgesic effects. The NLRP3 inflammasome can be activated by K+ efflux, Reactive oxygen species (ROS) releasing, and lysosomal disruption [29, 30]. Mitochondrial damage is the primary trigger of excessive ROS production and lysosomal dysfunction, which might induce obvious activation of NLRP3 inflammasome [31]. However, mitophagy may block the activation of NLRP3 inflammasome and subsequent of pain signal transmission [32, 33]. Autophagy is an adaptive response of eukaryotic cells to stressful stimuli wherein damaged cellular components are recycled via formation of autophagosomes. Selective elimination of damaged mitochondria through mycophagy in the microglia is crucial for neuron survival and transmission of pain signals [34]. Furthermore, mitophagy can inhibit NLRP3 inflammasome activation and reduce the secretion of IL-1β, most likely by preventing mitochondrial ROS production and reversing mitochondrial membrane depolarization [35]. Therefore, NLRP3 inflammasome inhibitors associated with mitophagy induction is worth investigating as analgesics against neuropathic pain. Gastrodia elata Blume is a traditional Chinese medicine used for the treatment of headache, migraine, and Shao et al. Journal of Neuroinflammation (2021) 18:142 other neuropathic pain, from which gastrodin was isolated as the main active constituents. Divanillyl sulfone (DS) is a derivative of 4,4′-dihydroxydibenzyl sulfoxide, which also was reported as the active constitute of Gastrodia elata Blume [36]. In the preliminary evaluation, DS showed potential pharmacological activity against neuropathic pain. In this study, we established somatic and neuropathic pain models to evaluate the antinociceptive effects of DS and explored the underlying mechanisms involving NLRP3 inflammasome activation in the microglia. Materials and methods Page 3 of 18 of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College. Establishment of somatic pain model and behavioral assessment Acetic acid-induced somatic pain model was established to preliminarily evaluate the analgesic effect of DS. The ICR mice were given a single intraperitoneal injection of DS (1, 3, or 10 mg/kg), normal saline (10 mL/kg), or gastrodin (positive control; 80 mg/kg), followed by 1% acetic acid (10 mL/kg, i.p.) 30 min later. The numbers of writhing and stretching in mice were counted over a period of 15 min after acetic acid injection. Drugs and reagents DS was synthesized and gastrodin was extracted and purified by the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, and the purity was validated as > 95% using high-performance liquid chromatography. Stock solution of both reagents were prepared in dimethyl sulfoxide (DMSO) and subsequently diluted in sterile 0.9% saline. The cell culture reagents were purchased from Invitrogen Corporation (Thermo Fisher Scientific, Carlsbad, CA, USA). Antibodies for Western blotting and immunofluorescence were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Alexa 546-conjugated goat anti-rabbit and Alexa 488-conjugated goat anti-mouse secondary antibodies were from Life Technologies (Thermo Fisher Scientific, Carlsbad, CA, USA). Goat serum, Mito-Tracker Green, Lyso-Tracker Red, and Rh123 were purchased from Beyotime Biotechnology (Shanghai, China). Nigericin, 3-methyladenine (3-MA), and MCC950 were purchased from MedChemExpress (Monmouth Junction, NJ); lipopolysaccharide (LPS) and tritonX-100 from Sigma-Aldrich (St. Louis, MO); and recombinant IL-1Ra from PeproTech (Rocky Hill, NJ, USA). CellROX® Deep Red Reagent was purchased from Invitrogen (Carlsbad, CA, USA), annexin-V/7aad staining kit from BD Biosciences (Franklin Lakes, NJ, USA), and FAM-FLICA® caspase assay kit from Immunochemistry Technologies (Bloomington, MN, USA). Experimental animals Male ICR mice and C57BL/6 mice (weighing 18–20 g) were obtained from Beijing Huafukang Experimental Animal Institute (Beijing, China). The mice were housed 5–6 per cage at room temperature (22 ± 2 °C) in specific pathogen-free conditions under a 12/12-h reversed lightdark cycle, with food and water provided ad libitum. The mice were acclimatized for 3–4 days prior to the experiments, and randomly divided into the different groups. All procedures were approved by the Experimental Animal Care and Use Committee of the Institute Establishment of neuropathic pain model and behavioral analysis Chronic neuropathic pain following peripheral nerve injury was simulated via chronic constrictive injury (CCI) of the unilateral sciatic nerve. Briefly, the C57BL/6 mice were anesthetized with isoflurane, and randomly divided into the sham-operated, untreated CCI model, DStreated (1, 3, and 10 mg/kg) and gastrodin (80 mg/kg) groups (n = 8 mice per group). The left sciatic nerve trunk was exposed by blunt dissection at mid-thigh level, and 4 ligatures (4-0 chromic catgut) were tied loosely around the nerve with 1 mm spacing. In the shamoperated mice, the sciatic nerve was only exposed but not ligated. On the ninth day after surgery, the mice were given a single intrathecal injection of the suitable dose of DS or saline. Briefly, the mice were anesthetized with isoflurane (4% for induction and 1% for maintenance), and a 100 μL micro-injector was inserted from the intervertebral space between the L5 and L6 discs into the spinal subarachnoid space. After confirming proper intrathecal injection via tail flicking, 100 μL normal saline or drug was microinjected followed by a 100 μL normal saline flush. The sensitivity of mechanical nociception was measured by the Von Frey withdrawal test (Von Frey filaments, IITC Life Science Inc, California, USA) after 30 min, 1 h, and 2 h of intrathecal injection over a period of 4 days. The animals were acclimatized in boxes set on an elevated metal mesh floor for at least 30 min. Pressure values were pressed vertically on the sole of the hind paws with increasing force till the animal withdraw the hind limb. The procedure was repeated 3 times for an average data, and the value of paw withdrawal threshold was recorded. All behavioral analyses were performed by an investigator blinded to the experimental grouping. The half-effective dose (ED50) of DS was determined by Von Frey withdrawal test on CCI-induced neuropathic pain model mice. Model mice were treated with a serious dose of DS (1, 3, 10, 30, 100 mg/mg) to test the Shao et al. Journal of Neuroinflammation (2021) 18:142 paw withdrawal threshold at 0.5 h, 1 h, and 2 h after administration. The values of peak time of paw withdrawal threshold were selected to calculate the ED50 of DS on CCI-induced model mice. Page 4 of 18 according to the manufacturer’s instructions. The optical density (OD) at 450 nm was obtained by an ELISA plate reader (Synergy H1, BioTek, USA). Immunofluorescence BV-2 cells culture and treatment BV-2 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2 and 95% humidity. The cells were pre-stimulated with LPS (100 ng/mL) for 3.5 h, and then with 5 μM nigericin for 30 min, 6 h, 12 h, and 24 h as appropriate. In addition, the cells were treated with varying concentrations of DS (0.3, 3, and 30 μM) or 3-MA (5 mM) for 1 h and 30 min respectively prior to nigericin. Extraction and separation of cell supernatant protein Cell supernatant protein was extracted by the standard methanol/chloroform method. The cell supernatants were layered with 1/4 volume of chloroform, and the same volume of methanol was added. After evenly mixing the solutions on a vortex shaker, the mixture was centrifuged at 12,000 rpm for 5 min at room temperature. The upper aqueous phase (methanol) was carefully removed, and the intermediate protein layer was aspirated and extracted once with methanol as described above. BV-2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.2% triton X-100 in phosphate buffered saline (PBS) containing 10% goat serum for 30 min. The cells were then incubated overnight with anti-NLRP3 (1:200) and antiASC (1:200) primary antibodies at 4 °C, washed with PBS, and probed with donkey anti-goat Alexa Fluor 488and goat anti-rabbit Alexa Fluor 546-conjugated secondary antibodies (1:200) for 1 h at 37 °C. The nuclei were counterstained with DAPI (1 μg/mL), washed thrice with PBS, and observed under the Leica TCS SP8 confocal microscope (Leica Microsystems GmbH, Mannheim, Germany). Spinal cord tissues from the CCI mice were also stained with anti-NLRP3 and anti-IBA-1 antibodies as described above. Mitochondrial and lysosomal imaging BV-2 cells were seeded in laser confocal plate (Nest, Jiangsu, China), and incubated with 75 nM Lyso-Tracker Red and 150 nM Mito-Tracker Green at 37 °C for 1 h. The nuclei were counterstained with DAPI (1 μg/mL), washed thrice with PBS, and observed under the Leica TCS SP8 confocal microscope. Western blotting analysis Proteins were quantified by BCA Protein Assay Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Equal amount of proteins per sample were separated by 8% and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the bands were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp., Bedford, MA, USA). After blocking with 5% bovine serum albumin (BSA) in tris-based saline-Tween 20 (TBST) at room temperature for 1 h, the blots were incubated overnight with primary antibodies against IBA-1, NLRP3, pro-caspase-1, caspase-1, pro-IL-1β, IL1β, LC3II/I, Beclin 1, protein 62 (p62), and β-tubulin (all diluted 1:1000) at 4 °C. Following incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies, the blots were developed using enhanced chemiluminescence reagents (Perkinelmer, USA). The positive bands were visualized with Tanon 2000 Imaging System (Beijing, China) and their intensities were quantified using ImageJ Software (NIH, USA). Enzyme-linked immunosorbent assay The level of IL-1β in the supernatant from cultured BV2 cells was measured by enzyme-linked immunosorbent assay (ELISA) kit (Cat. No. 432606, BioLegend) Mitochondrial membrane potential and reactive oxygen species measurement Mitochondrial membrane potential (MMP) was measured using the cationic fluorescent probe Rh123 which rapidly translocated from the mitochondrial membrane to the matrix following membrane depolarization. The reactive oxygen species (ROS) levels were detected using CellROX Deep Red, which emits a strong fluorescence signal under oxidizing conditions. The suitably treated cells were incubated with 10 μM Rh123 or 5 μM CellROX Deep Red at room temperature for 30 min, and 1 μg/mL DAPI was added 10 min before the end of staining. After washing with PBS, the cells were observed under the Leica TCS SP8 confocal microscope to determine the fluorescence intensities of the respective probes. Flow cytometry BV-2 cells were cultured and prepared to detect by flow cytometric staining with propidium iodide (PI)-PerCP and caspase-1-FITC (FAM-YVAD-FMK, Immunochemistry Technologies) for NLRP3 inflammasome activation; PI-PerCP and caspase-3/7-FITC (FAM-DEVD-FMK, Immunochemistry Technologies), and 7aad-PerCP and Annexin-V-APC for apoptosis (BD Biosciences). Briefly, Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 5 of 18 the cultured cells at 37 °C were stimulated by LPS (100 ng/mL) for 3.5 h, incubation with or without 3-MA (5 mM) for another 30 min, and then incubation with DS (0.3, 3, and 30 μM for apoptosis measurement; 30 μM for NLRP3 inflammasome activation measurement) for 1 h. After stimulating with nigericin (5 μM), the harvested cells were washed twice with ice-cold PBS, and stained with 7aad or PI for 5 min at room temperature, Annexin-V for 15 min at room temperature, or FAMYVAD-FMK (caspase-1) and FAM-DEVD-FMK (caspase-3/7) for 1 h at 37 °C, respectively. The samples were acquired in a FACS BD verse cytometer (BD Biosciences), and data were analyzed using FlowJo software (Version 10.0; Three Star). compared by two-factor analysis of variance (ANOVA) followed by Tukey’s post hoc test. The percentage of analgesia was calculated using the formula: (post-DS threshold in ipsilateral paw − baseline threshold in ipsilateral paw) / (baseline threshold in contralateral paw − baseline threshold in ipsilateral paw) × 100. Analysis of ED50 was performed with GraphPad best projected by the non-linear least-squares method in which data is normalized according to the maximal possible effect. The other parameters were analyzed using one-way ANOVA followed by an appropriate post hoc test, and P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 8.01 software (GraphPad Software Inc., CA, USA). Statistical analysis Results Data were presented as the mean ± SEM of at least 3 independent experiments. The paw withdrawal threshold (PWT (g)) was measured with Von Frey test and DS attenuated somatic pain in vivo DS, 4,4′-[Sulfonyl bis(methylene)] bis(2-methoxyphenol) (Fig. 1a), is obtained by acetic acid/hydrogen peroxide Fig. 1 DS attenuated pain hypersensitivity of somatic pain in vivo. a The chemical structure of DS. DS significantly decreased the writhing times (b) and increased the rate of analgesia (c) in acetic acid-induced mice. Data are expressed as mean ± SEM (n = 9 mice in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where P < 0.01, P < 0.001 vs. saline group Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 6 of 18 oxidation of 4,4′-[sulfinyl bis(methylene)] diphenol. We established a mouse model of somatic pain by injecting ICR mice with low concentration of acetic acid (1%), and found that intraperitoneal administration of DS significantly reduced the number of writhing behavior compared to the placebo in a dose-dependent manner. In addition, the analgesic effect of 1, 3, and 10 mg/kg DS were respectively 21.00%, 72.48%, and 74.53% compared to 48.48% observed with 80 mg/kg gastrodin (Fig. 1b, c). antagonist (IL-1Ra). As shown in Fig. 3d, e, intrathecal injection of MCC950 and IL-1Ra significantly increased the mechanical withdrawal threshold, and the analgesic effect was stable for four consecutive days after administration. Taken together, IL-1β secreted by the activated NLRP3 inflammasome relays pain signals by binding to the IL-1R expressed on neurons, and the analgesic effect of DS may be related to blocking NLRP3 inflammasome activation and IL-1β/IL-1R signaling in the spinal cord. DS alleviated CCI-induced neuropathic pain in vivo DS inhibited NLRP3 inflammasome activation by promoting autophagic flux formation A mouse model of chronic neuropathic pain was established by sciatic nerve ligation, and the effect of DS on mechanical allodynia was evaluated in terms of paw withdrawal. Sciatic nerve ligation decreased the paw withdrawal threshold of the ipsilateral but not the contralateral paw, and the threshold reached its lowest point on the seventh day post-surgery and maintained at this value for the rest of testing period, indicating that the neuropathic pain model was successfully induced (Fig. 2a). However, intrathecal injection of DS increased paw withdrawal threshold in the ipsilateral paw in a dose-dependent manner compared to the placebotreated mice, without affecting the contralateral paw. The analgesic effect of DS peaked within 30 min of injection in a dose-dependent manner, whereas 80 mg/kg gastrodin achieved the optimal effect 1 h after intrathecal injection (Fig. 2b). The analgesic effects of both drugs were stable over four consecutive days post-DS injection (Fig. 2c). In addition, further dose-response analyses showed that DS alleviated allodynia in ipsilateral paw in a dose-dependent manner, with a ED50 values of 5.29 mg/kg at 0.5 h after administration DS (Fig. 2d, Figure S1). DS inhibited NLRP3 inflammasome activation in the spinal microglia in vivo The glial area in the ipsilateral enlarged lumbar spinal cord expressed high levels of the microglial activation marker Iba-1 after sciatic nerve ligation, whereas the contralateral spinal cord was not affected. This clearly indicated spinal microglial activation in the CCI pain model (Fig. 3a). In addition, the activated Iba-1+ microglia co-localized with the NLRP3 protein, indicating that neuropathic pain induced by microglial activation is likely related to the activation of NLRP3 inflammasome (Fig. 3a). DS not only inhibited the co-localization of NLRP3 and IBA-1 in spinal microglia but also decreased the in situ expression of IBA-1, NLRP3 protein, activated caspase-1 p10, and mature IL-1β p17 (Fig. 3b, c). To further explore the role of NLRP3 inflammasome in neuropathic pain, we respectively treated the CCI model mice with MCC950, a specific inhibitor of the NLRP3 inflammasome, and the recombinant IL-1R The in vitro model of NLRP3 inflammasome activation was established by co-stimulating BV-2 microglia with LPS and nigericin. The cells were first exposed to LPS to upregulate the NLRP3 inflammasome components, followed by DS and finally nigericin. DS treatment significantly reduced the expression of NLRP3 in cell lysates (LYS), as well as maturation of caspase-1 and IL1β in the cultured supernatants (SN), in response to LPS and nigericin (Fig. 4a, b). Similarly, DS also inhibited the nigericin-induced secretion of IL-1β in a dosedependent manner (Fig. 4c). These findings were similar to that observed in the spinal cord tissues of DS-treated CCI mice. Autophagy flux is a chain process from autophagosome formation to ubiquitin degradation through fusion with lysosomes. Among them, mitophagy play an important role in the phagocytosis of damaged mitochondria. Given the inhibitory role of mitophagy in NLRP3 activation, we next determined whether the therapeutic effects of DS were mediated via mitophagyinduced NLRP3 inflammasome inhibition. Microtubuleassociated light chain protein 3 (LC3) is a necessary autophagy marker for the formation of autophagy membrane. In addition, p62 protein is also responsible for the process of autophagy by interacting with LC3 to bring damaged mitochondria and other autophagosomes into autolysosomes for degradation and self-degradation. The results indicated that DS upregulated the expression of LC3II and Beclin 1, increased the ratio of LC3II/LC3I, and decreased the expression of p62 protein and the level of IL-1β in the cultured supernatants (Fig. 4d–f). These data suggested that DS likely promoted mitophagy in the microglia. To better understand the mechanism of increased mitophagy and negative regulation of NLRP3 inflammasome by DS, we also measured the expression of autophagy-associated protein and NLRP3 in the presence of autophagy inhibitor 3-MA. The findings indicated that 3-MA not only blocked the effect of DS on autophagy-related proteins, but also restored NLRP3 activation by increasing the level of IL-1β (Fig. 4d–f). Furthermore, mito-tracker and lyso-tracker were used to trace the formation of autolysosomes during mitophagy in BV-2 microglia. The results indicated that Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 7 of 18 Fig. 2 DS treatment improved sciatic nerve ligation-induced chronic neuropathic pain. Neuropathic pain mice were induced by ligation of sciatic nerve, the mechanical withdrawal threshold within 14 days after surgery (a) and intrathecal injection of vehicle (0.9% normal saline) or DS (1, 3, 10 mg/kg) 9 days after surgery (b) were measured by Von Frey Monofilament in both contralateral and ipsilateral paws. c The analgesic effects of both drugs over four consecutive days post-injection. d Dose-response analysis of DS on paw withdrawal threshold 0.5 h after administration, and ED50 was calculated with GraphPad Prism 8 with normalized to maximal possible effect. Data are expressed as means ± SEM (n = 8 mice in each group). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc analysis where P < 0.05, P < 0.01, P < 0.001 vs. CCI + Saline group, *P < 0.001 vs. Sham-ipsilateral paw group mitochondria damaged by LPS and nigericin were transferred to lysosome for degradation in the process of DSinduced mitophagy, which was also inhibited by the autophagy inhibitor 3-MA (Fig. 4g). These findings suggested that DS promoted the fusion of mitochondria and lysosomes. To further verify whether DS inhibited the activation of NLRP3 inflammasome through mitophagy, we analyzed the expression levels of individual components of NLRP3 inflammasome in the presence of autophagy inhibitor 3-MA. Our findings showed that DS inhibited the expression of NLRP3 in LYS as well as the secretion of mature caspase-1 p10 and IL-1β p17 in supernatants in the presence of LPS and nigericin, and which could be completely neutralized by 3-MA (Fig. 5a). In addition, our results also indicated that LPS and nigericin induced an obvious overexpression of NLRP3 and ASC in cultured BV-2 microglia, which could be blocked by DS treatment (Fig. 5b). However, 3-MA could reverse the inhibitory effect of DS on the overexpression of NLRP3 and ASC. Furthermore, LPS and nigericin stimulated caspase-1 activation and the conversion from procaspase-1 to caspase-1, which appeared as an increase in the population of double-positive cells stained with Caspase-1 and Propidium Iodide (PI) (Fig. 5c, d). Meanwhile, DS obviously inhibited the caspase-1 activity in BV-2 microglia, and this effect could be neutralized by 3-MA (Fig. 5c, d). Taken together, mitochondrial damage induced by LPS and nigericin triggered NLRP3 inflammasome activation. However, DS inhibited Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 8 of 18 Fig. 3 DS showed analgesic effect by inhibiting NLRP3 inflammasome activation of spinal microglia in vivo. a The immunofluorescence of the spinal lumbar enlargement of CCI pain mice showed the co-localization of IBA-1 staining (red) and NLRP3 staining (green) in the ipsilateral glial area, and the contralateral side was not affected. b, c Western blotting analysis of microglia activation marker IBA-1 and NLRP3 inflammasome components in spinal lumbar enlargement tissue showed that DS inhibited the expression of IBA-1, NLRP3 protein, activated caspase-1 p10, and mature IL-1β p17 in a dose-dependent manner. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ##P < 0.01, ###P < 0.011 vs. Sham + Saline group; P < 0.05, P < 0.01, P < 0.001 vs. CCI + Saline group. NLRP3 inflammasome inhibitor MCC950 (d) and IL-1R antagonist (e) were injected intrathecally into CCI pain mice and the paw withdrawal threshold of their ipsilateral and contralateral paws were measured at 1h post intrathecal injection. Inhibiting the assembly and activation of NLRP3 inflammasome or block the binding of IL-1β to IL-1R can effectively increase the paw withdrawal threshold of CCI mice. Data are expressed as means ± SEM (n = 8 in each group). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc analysis where P < 0.01, *P < 0.001 vs. CCI + Saline group Shao et al. Journal of Neuroinflammation Fig. 4 (See legend on next page.) (2021) 18:142 Page 9 of 18 Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 10 of 18 (See figure on previous page.) Fig. 4 DS inhibited NLRP3 inflammasome activation and promoted mitophagy in microglia in vitro. BV-2 microglia were first pre-stimulated by LPS (100 ng/ml) for 3.5 h, then treated for 1h with indicated concentrations of DS followed by stimulation with nigericin (5 μM) for 30 min. a, b Immunoblot analyses of caspase-1 p10, IL-1β p17 in culture supernatants (SN) and NLRP3, pro-caspase-1, pro-IL-1β in lysates (LYS) were shown. c The level of IL-1β in supernatants was analyzed by ELISA method. After pre-stimulated with LPS, adding autophagy inhibitor 3-MA (5 mM) before DS and incubated for 0.5 h, and finally stimulated with nigericin for 12 h. d, e Immunoblot analyses of NLRP3, LC3II/I, Beclin 1 and p62 in LYS and c ELISA analysis of IL-1β in SN. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ##P < 0.01, ###P < 0.001 vs. control; P < 0.05, P < 0.01, P < 0.001 vs. LPS + Nigericin group; $P < 0.05, $$P < 0.01, vs. LPS + Nigericin + 30 μM DS group. e Mitochondria and lysosome were respectively labeled with the mito-tracker (green) and lyso-tracker (red), images were captured using Leica TCS SP8 confocal microscope formation of NLRP3 inflammasome complex in BV-2 microglia, which was manifested by a decrease in both caspase-1 activity and IL-1β secretion, by inducing mitophagy and promoting clearance of defective mitochondria. DS decreased ROS production and inhibited apoptosis in microglia by inducing mitophagy Although our findings indicate that mitophagy induced by DS inhibited the activation of NLRP3 inflammasome in microglia, the underlying mechanism is still unclear. As an important signal to promote the activation of inflammasome, ROS may be involved in the negative regulation of NLRP3 by mitophagy. Therefore, in order to explore whether the analgesic effect of DS was related to autophagic clearance of ROS to inhibit NLRP3 inflammasome activation, the changes of mitochondrial function in microglia with the treatment of DS were assessed. The effect of DS on mitochondrial function Fig. 5 DS negatively regulated the formation of NLRP3 inflammasome complex through mitophagy in BV-2 microglia. BV-2 microglia were first pre-stimulated by LPS (100 ng/ml) for 3.5 h, then treated for 1h with indicated concentrations of DS and 3-MA (5 mM) for 30 min or not followed by stimulation with nigericin (5 μM) for 6 h, 12 h, and 24 h, which was labeled with a triangle at nigericin (5 μM) treatment. a Immunoblot analyses of caspase-1 p10, IL-1β p17 in culture supernatants (SN) and NLRP3, pro-caspase-1, pro-IL-1β in lysates (LYS) were shown. b Immunofluorescence staining show the expression of NLRP3 (green) and ASC (Red) in BV-2 microglia after stimulation with nigericin for 12 h. c, d Caspase-1 activity was detected by flow cytometry in BV-2 microglia treated with DS for 12 h using caspase-1/PI double-labeled staining method. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ###P < 0.001 vs. control group; *P < 0.001 vs. LPS + Nigericin group; $$$P < 0.001 vs. LPS + Nigericin + 30 μM DS group Shao et al. Journal of Neuroinflammation (2021) 18:142 was investigated by measuring ROS production and MMP using specific fluorescent probes. As shown in Fig. 6a, NLRP3 inflammasome activation in response to LPS and nigericin significantly increased the fluorescence intensities of both Rh123 and CellROX Deep Red, indicating a high level of mitochondrial membrane depolarization and ROS production respectively. DS dampened the fluorescence intensities of both probes, which was suggestive of MMP restoration and reduction in intracellular ROS levels. Furthermore, 3-MA could counteract the protective effects of DS, indicating that DS promoted ROS clearance in the microglia through mitophagy. In addition, damaged mitochondria release DAMPs through apoptosis, resulting in a vicious circle of NLRP3 inflammasome activation and mitochondrial dysfunction, and irreversible tissue damage. Therefore, we analyzed the effect of DS on apoptosis to elucidate the potential mechanism underlying NLRP3 inflammasome inactivation. Bax and Bcl-XL are two important proteins involved in the process of apoptosis program. Our research found that DS upregulated the expression of anti-apoptotic Bcl-XL and inhibited the expression of pro-apoptotic Bax in the stimulation of LPS and nigericin which were responsible for formation of NLRP3 inflammasome complex. However, 3-MA could reverse these changes (Fig. 6b, c). Elevated caspase-3/7 activity is an important indicator in the process of apoptosis program. In our study, LPS and nigericin increased the activity of caspase-3/7 and percentage of apoptotic cells in cultured BV-2 microglia. However, DS could block the apoptosis (Fig. 7a, b) and caspase-3/7 activity (Fig. 7c, d) in a time and concentration-dependent manner, which was completely obviated by 3-MA. Taken together, DS promoted mitophagy induced ROS clearance and inhibited apoptosis in activated microglia, which may be responsible for inhibition of NLRP3 inflammasome complex. DS ameliorated neuropathic pain through mitophagymediated NLRP3 inflammasome inhibition To further explore the possible mechanism DS exerting analgesic effect, autophagy inhibitor 3-MA was applied in our research trying to clarify the regulation of mitophagy on NLRP3 inflammasome. On day nine of the CCI pain model, 3-MA was administrated by intrathecal injection 30 min before the administration DS, and the bilateral paw withdrawal threshold of each group of CCI mice were measured at 30 min, 1 h, and 2 h after administration DS. The results showed that DS could obviously increase the pain threshold of CCI mice and reach the peak of analgesia 30 min after administration DS. However, the autophagy inhibitor 3-MA could block the analgesic effect of DS (Fig. 8a). After four consecutive days of same administration, it was found that the analgesic Page 11 of 18 effect of DS was relatively stable and no obvious tolerance occurred (Fig. 8b). In addition, four consecutive days of 3-MA treatment not only reversed the effect of DS, it even lowered the pain threshold than that of the model group (Fig. 8b), which suggested that mitophagy played an important role in the occurrence and development of neuralgia, and DS might exert an analgesic effect by inducing mitophagy. Further analysis by Western blot found that the inhibitory effect of DS on the activation of NLRP3 inflammasome in the spinal cord tissue of CCI mice was countered by 3-MA (Fig. 8c, d), indicating that mitophagy played a negative regulatory role on the activation of NLRP3 inflammasome in CCI-induced pain model. These data indicated that DS may inhibit the activation of NLRP3 inflammasome signaling via inducing mitophagy in CCI-induced neuropathic mice, thereby reducing the pain signal conduction caused by the binding of IL-1β to IL-1R on the spinal dorsal horn neurons, therefore raising the pain threshold. Discussion Microglia account for 5–10% of all cells in the CNS and play an important role in innate immune responses. However, aberrantly activated microglia can trigger neuroinflammation and tissue damage [18, 37]. Peripheral and central nerve damage in animal models of neuropathic pain are accompanied by the upregulation of inflammatory factors, chemokines, brain-derived neurotrophic factor (BDNF), toll-like receptors (TLRs), and purinergic receptor in the microglia [38, 39]. Thus, aberrant phenotypic and functional changes in microglia accompany the initiation and development of neuropathic pain, which involve NLRP3 inflammasome activation [14, 40]. PNI induces a release of PAMPs and DAMPs in glial region of the spinal cord, which are recognized by the microglia. PAMPs activate the priming signal of NLRP3 inflammasome, and DAMPs act as the second signal that promote neuroinflammation and facilitate transmission of pain signals from the primary center to the superior center [11, 14, 20, 21]. Blocking NLRP3 inflammasome activation in spinal cord microglia could suppress PNI-induced pain hypersensitivity [41, 42]. Mitophagy is a conserved defense mechanism in eukaryotic cells, and has been implicated in the inhibition of NLRP3 inflammasome activation and neuropathic pain [29, 43, 44]. However, the exact mechanistic basis is not completely known. In this study, we assessed the analgesic properties of DS, derivative from an active constitute of the traditional Chinese medicine Gastrodia, and established the functional link between mitophagymediated inhibition of NLRP3 inflammasome activation and alleviation of neuropathic pain. Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 12 of 18 Fig. 6 DS promoted autophagic clearance of mitochondrial ROS and protected MMP through mitophagy in microglia. BV-2 microglia were first pre-stimulated by LPS (100 ng/ml) for 3.5 h, then treated for 1 h with indicated concentrations of DS and 3-MA (5 mM) for 30 min or not followed by stimulation with nigericin (5 μM) for 12 h. a Immunofluorescence staining Rh123 (green) and CellROX Deep Red (Red) indicate mitochondrial membrane depolarization and ROS production in BV-2 microglia. b, c Immunoblot analyses the expression of apoptosis associated proteins of Bcl-XL and Bax in lysates. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by oneway ANOVA followed by Tukey’s post hoc analysis where ##P < 0.01 vs. control group; P < 0.05, *P < 0.01 vs. LPS + Nigericin group; $P < 0.05 vs. LPS + Nigericin + 30 μM DS group Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 13 of 18 Fig. 7 DS inhibited apoptosis and caspase-3/7 activity through mitophagy in BV-2 microglia. BV-2 microglia were first pre-stimulated by LPS (100 ng/ ml) for 3.5 h, then treated for 1 h with indicated concentrations of DS and 3-MA (5 mM) for 30 min or not followed by stimulation with nigericin (5 μM) for 12 h. Apoptosis was detected by flow cytometry in BV-2 microglia treated with indicated concentrations of DS for 6, 12, and 24 h using Annexin V-FITC/7aad (a, b) and caspase-3/7/PI (c, d) double-labeled staining method. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; P < 0.05, P < 0.01, P < 0.001 vs. LPS + Nigericin group; $P < 0.05, $$P < 0.01, $$$P < 0.001 vs. LPS + Nigericin + 30 μM DS group Shao et al. Journal of Neuroinflammation (2021) 18:142 Page 14 of 18 Fig. 8 DS ameliorated neuropathic pain through mitophagy-mediated NLRP3 inflammasome inhibition. a Paw withdrawal threshold was measured by Von Frey test in both contralateral and ipsilateral paws, after administrating DS (10 mg/kg), DS (10 mg/kg) + 3-MA (2.5 mg/kg), and vehicle (0.9% normal saline) 9 days in CCI-induced model mice, respectively. b The analgesic effects of both DS (10 mg/kg) and DS (10 mg/kg) + 3-MA (2.5 mg/kg) treatment over four consecutive days post-injection. Data are expressed as means ± SEM (n = 7 mice in each group). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc analysis where P < 0.01 vs. CCI + Saline group, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CCI + 10 mg/kg DS group. c, d Western blotting analysis of NLRP3 inflammasome components in spinal lumbar enlargement tissue. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where #P < 0.05, ##P < 0.01 vs. Sham + Saline group; P < 0.05 vs. CCI + Saline group; $P < 0.05 vs. CCI + 10 mg/kg DS group Microglia acts as a kind of typical brain-resident macrophages to phagocytose the damaged debris in CNS. During the chronic neuropathic pain injury, microglia is activated and polarized into pro-inflammatory phenotype and chronic neuroinflammation [45]. In the CCI pain model as well, sciatic nerve ligation increased IBA1-positive microglia in the spinal lumbar area; this is accompanied by a gradual decrease in the mechanical pain threshold on the ligated side. It has been reported that activated microglia co-localized with NLRP3 protein, indicating NLRP3 inflammasome activation in the microglia [10, 25]. The central sensitization and pain development caused by microglial activation are related to the activation of NLRP3 inflammasome. For example, the NLRP3 inflammasome inhibitor MCC950 could increase the pain threshold of CCI mice. In addition, the NLRP3 inflammasome effector IL-1β is an important pro-inflammatory factor that promotes pain signal transduction [18, 26, 46]. IL-1Ra inhibited the IL-1β/IL-1R signaling and increased the pain threshold of CCI mice, further underscoring the role of NLRP3 inflammasome in neuralgia. It was found that DS significantly alleviated the neuropathic pain by increasing mechanical withdrawal threshold in CCI-induced neuropathic pain mice. Meanwhile, DS effectively inhibited the activation of NLRP3 inflammasome in LPS and nigericin-stimulated Shao et al. Journal of Neuroinflammation (2021) 18:142 BV-2 microglia cells by suppressing caspase-1 activation and IL-1β secretion. Given that DS affects the expression of NLRP3 inflammasome-associated proteins in activated BV-2 cells and CCI-induced neuropathic pain model mice, as well as DS or NLRP3 inflammasome inhibitor MCC950 and IL-1Ra all can ameliorate the neuropathic pain by increasing the mechanical withdrawal threshold, it is suggested that DS reduce the nociception likely via NLRP3 inflammasome mechanism. Thus, our work hints that focus on inhibiting NLRP3 inflammasome activation might be a new therapeutic strategy for chronic neuropathic pain. Increasing evidence suggests that damaged mitochondria involved in the progress of disease with stress, ROS production, and NLRP3 inflammasome activation, including chronic neuropathic pain. An important role of mitophagy is to selectively remove excess or damaged mitochondria. Following ROS-induced depolarization, the damaged mitochondria are engulfed by the Page 15 of 18 autophagosomes that fuse with the lysosomes, eventually reducing intracellular ROS levels and inhibiting NLRP3 inflammasome activation [47, 48]. During mitophagy, the endoplasmic reticulum membrane wraps the damaged mitochondria to form autophagosomes, and the subsequent modification of the LC3-I protein to LC3II protein promotes their fusion with lysosomes [49, 50]. The autophagic flux is also driven by degradation of the ubiquitin-binding protein p62 [51], and the dissociation of Beclin-1 from the anti-apoptotic Bcl-2 or Bcl-XL [52]. Beclin-1 cleavage by caspases on the other hand inhibits autophagy and triggers the apoptotic cascade by promoting cytochrome C release from mitochondria [53, 54]. In this study, we found that DS alleviated the mitochondrial damage and ROS generation induced by mitochondrial membrane depolarization in LPS and nigericin-stimulated microglia by promoting mitophagy. Therefore, our research further confirmed that mitophagy contributed to the Fig. 9 Proposed analgesic mechanism of DS for mitophagy-induced NLRP3 inflammasome inhibition in spinal microglia Shao et al. Journal of Neuroinflammation (2021) 18:142 process of mitochondrial protection and ROS clearance in excessive activated microglia. Mitophagy blockade is often accompanied by severe mitochondrial damage and ROS production, and the subsequent activation of NLRP3 inflammasome [55, 56]. Therefore, the autophagic flux is a potential therapeutic target in NLRP3 inflammasome-related diseases. Chronic neuropathic pain is characterized by mitochondrial dysfunction and accumulation of ROS in the glial area of the spinal cord, which act as DAMPs for the NLRP3 inflammasome activation. Notably, autophagy increases mechanical pain threshold in neuropathic pain models, likely by inhibiting NLRP3 inflammasome activation [40, 57]. However, the mechanism underlying the regulatory role of mitophagy-mediated NLRP3 inflammasome on chronic neuropathic pain remain poorly understood. Here, our research indicated that DS induced an obvious mitophagy and ROS reduction in cultured microglia. To determine the role of mitophagy on NLRP3 inflammasome activation, the mitophagy/autophagy inhibitor 3-MA was used to investigate the regulation for NLRP3 inflammasome under DS treatment. The results showed that 3-MA neutralized DSinduced mitophagy in the activated microglial cells, which hindered degradation of damaged mitochondria and removal of ROS. Moreover, DS-mediated inhibition of NLRP3 inflammasome activation and mature IL-1β secretion from the microglia were also blocked by 3-MA. In vivo experiment further also verified that 3-MA could prevent the analgesic effect of DS in CCI-induced neuropathic pain model mice. Therefore, these data indicated that DS-induced mitophagy is accompanied by inactivation of NLRP3 inflammasome. Briefly, the current findings suggested that the effect of DS on chronic neuropathic pain may be associated with mitophagy induction, which mediates the NLRP3 inflammasome inactivation and neuroinflammatory inhibition. Conclusion In summary, we reported that DS effectively improved the chronic neuropathic pain in CCI-induced neuropathic pain model mice. The underlying mechanisms have been investigated that DS promoted rapid clearance of ROS released by dysfunctional mitochondria through mitophagy, and further suppressed the NLRP3 inflammasome activation and neuroinflammation (Fig. 9). Our work has revealed the negative regulation of mitophagy to NLRP3 inflammasome in neuropathic pain, which suggested that NLRP3 inflammasome may be a promising therapeutic strategy for chronic neuropathic pain. The findings also provide a potential candidate for treatment of chronic neuropathic pain. Page 16 of 18 Abbreviations DS: Divanillyl sulfone; IL-1β: Interleukin-1β; IL-18: Interleukin-18; IL1R: Interleukin-1 receptor; IL-1Ra: Interleukin-1 receptor antagonist; NLRP3: NLR family pyrin domain-containing 3; CNS: Central nervous system; DRG: Dorsal root ganglia; PRRs: Pattern recognition receptors; PAMPs: Pathogen-associated molecular patterns; DAMPs: Danger-associated molecular patterns; SDH: Spinal dorsal horn; NMDAR: N-methyl-D-aspartate receptor; GABA: γ-Aminobutyrate; LPS: Lipopolysaccharide; 3-MA: 3Methyladenine; CCI: Chronic constrictive injury; SN: Supernatants; LYS: Lysates; PBS: Phosphate buffer saline; MMP: Mitochondrial membrane potential; ROS: Reactive oxygen species; LC3: Microtubule-associated light chain protein 3; p62: Protein 62 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12974-021-02178-z. Additional file 1 . Acknowledgements Not applicable. Authors’ contributions SS and TTZ designed the research study and wrote the paper. XCB, GQL, and SJG isolated and synthesize the compound. SS, CCJ, SGN and ZY performed the experiment. WYZ and WL analyzed the data. The authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (No. 81973338), the National Key R&D Program of China (2020YFA0908004), Drug Innovation Major Project of China, China (No. 2018ZX09711001-011-005), and CAMS Innovation Fund for Medical Science, China (No. 2017-I2M-3-011). Availability of data and materials The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate Not applicable. All animals were treated in accordance with the International Guidelines for animal research. 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https://openalex.org/W2154234678	https://europepmc.org/articles/pmc4222069?pdf=render	English	null	HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase	Orphanet journal of rare diseases	2,013	cc-by	6,850	* Correspondence: shamima.rahman@ucl.ac.uk 4Metabolic Unit, Great Ormond Street Hospital, London, UK 6Mitochondrial Research Group, Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK Full list of author information is available at the end of the article © 2013 Ferdinandusse 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. Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase Sacha Ferdinandusse1, Hans R Waterham1, Simon JR Heales2,5,6, Garry K Brown7, Iain P Hargreaves5, Jan-Willem Taanman8, Roxana Gunny3, Lara Abulhoul4, Ronald JA Wanders1, Peter T Clayton6, James V Leonard6 and Shamima Rahman4,6* RESEARCH Open Access HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase Patient 2 A younger sister of Patient 1 is currently well at 9 years of age but the third child in the family, Patient 2, was a boy born at term after a normal pregnancy, with a birth weight of 2.9 kg. He presented on day 1 of life with poor sucking and feeding difficulties. Feeding problems continued and were associated with poor weight gain, necessitating nasogastric tube feeding from 2 months. Echocardiogram and ophthalmological examination were normal at 4 months. He subsequently developed infantile spasms, and hypsarrhythmia was observed on the electro- encephalogram (EEG) at 7 months. The infantile spasms responded dramatically to high dose prednisolone therapy. He also had a progressive dystonic disorder and persistent vomiting despite fundoplication (performed at 10 months), associated with abdominal pain, extreme irritability, sleep disturbance and breath-holding episodes leading to severe oxygen desaturation. Venous blood lactate was elevated at 4 mmol/l on one occasion but normal at other times (Table 1). CSF lactate was mildly elevated on two occasions at 2.1 and 2.6 mmol/l. The most significant biochemical ab- normality was persistent elevation of hydroxy-C4-carnitine (0.77 – 1.25 μmol/L, reference range <0.4; Figure 1b), an investigation which was not available when Patient 1 was alive. Normal biochemical investigations included very long chain fatty acids, transferrin electrophoresis, purines, creatine kinase, cholesterol and triglycerides, CSF neurotransmitters, urinary glycosaminoglycans and white cell enzyme assays for lysosomal storage disorders. Muscle biopsy showed mildly increased lipid deposition within muscle fibres but no ragged-red or COX-negative fibres. MRI brain at 11 months showed abnormal signal within the dentate nuclei and the globi pallidi, with a generalised lack of white matter (see Figure 2a and legend for details of imaging appearances). Relentless progres- sion led to death at 2 years 8 months. Neurological features of cerebral organic acidurias (disorders of degradation of the carbon skeleton of amino acids) can be clinically and radiologically indis- tinguishable from mitochondrial encephalomyopathies caused by primary RC deficiencies; seizures, neurological regression and bilateral symmetrical basal ganglia lesions may occur in both groups of disorders [7-10]. 3-Hydroxy- isobutyryl-CoA hydrolase (HIBCH) is a mitochondrial enzyme that catalyses the fifth step of valine catabolism, the conversion of 3-hydroxy-isobutyryl-CoA to 3-hydroxy- isobutyrate (Figure 1a). HIBCH deficiency has previously been reported in only two patients [11,12]. We now describe two new genetically confirmed cases (siblings), one of whom presented with combined defects of multiple RC enzymes and the pyruvate dehydrogenase complex (PDHc). Background vomiting. From 8 months he developed myoclonic jerks and from 10 months recurrent generalised seizures. Cerebrospinal fluid (CSF) lactate was mildly elevated at 2.2 mmol/l (reference range <2 mmol/l) at 8 months, with a normal venous blood lactate of 1.7 mmol/l (reference range < 1.8 mmol/l). A repeat CSF lactate was 3.5 mmol/l. Histological examination of an open muscle biopsy per- formed at 17 months revealed a mild increase in neutral lipid droplets, but no ragged-red or cytochrome oxidase (COX)-negative fibres. His clinical course was marked by persistent vomiting and retching, despite the fundoplica- tion, and extreme irritability and sleep disturbance. MRI brain showed altered signal and atrophy in the globi pallidi and relative sparing of the thalami. Leukoencephalopathy and some generalised atrophy were also observed. He died at 3 years of age. g Mitochondrial disorders affect approximately 1 in 5000 births, and are clinically, biochemically and genetically heterogeneous [1]. Combined deficiency of multiple respiratory chain (RC) enzymes is one of the most frequent findings in children with suspected mitochondrial disease, representing approximately 30% of cases in whom a biochemical abnormality is identified. Approximately 50% of patients with multiple RC deficiencies have impaired replication or maintenance of the mitochon- drial DNA (mtDNA), leading to progressive depletion of mtDNA [2] or accumulation of multiple mtDNA deletions. The remaining ~50% of cases have heteroge- neous underlying causes, including mitochondrial or nuclear-encoded defects of mitochondrial protein syn- thesis [3] and the multiple mitochondrial dysfunctions syndrome, in which the activity of PDHc is also impaired [4-6]. Defects in mtDNA repair, maintenance or trans- lation result in combined deficiency of complexes I, III and IV (i.e. complexes that contain mtDNA-encoded subunits) whereas the multiple mitochondrial dysfunc- tions syndrome usually affects complexes containing iron-sulphur (Fe-S) clusters (complexes I, II and III) as well as PDHc. Abstract Background: Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome. Methods: Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis. Results: Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G>A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family. Conclusions: HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency. Keywords: Mitochondrial disease, Multiple respiratory chain enzyme deficiencies, Pyruvate dehydrogenase deficiency, 3-hydroxy-isobutyryl-CoA hydrolase, HIBCH, Acylcarnitines, Multiple mitochondrial dysfunctions syndrome, Valine catabolism, Organic aciduria Page 2 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Patient 2 This potentially represents a new disease mech- anism mimicking the multiple mitochondrial dysfunctions syndrome, namely degradation of multiple enzymes result- ing from accumulation of a toxic metabolite methacrylyl- CoA that is postulated to reduce mitochondrial enzyme activities by reacting with exposed thiol groups. Patient 1 The index case was the first child of healthy distantly related Pakistani parents. He was born at term weighing 3.2 kg. There were no neonatal problems, but from 3 months he had developmental regression, with loss of smile and progressive hypotonia. At 8 months Nissen fundoplication was performed because of persistent Page 3 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Figure 1 HIBCH deficiency leads to accumulation of hydroxy-C4-carnitine. (a) Valine degradation pathway. 3-Hydroxy-isobutyrylCoA hydrolase (HIBCH) catalyses the fifth step of valine catabolism. HIBCH deficiency leads to accumulation of 3-hydroxy-isobutyryl carnitine, which is detected as hydroxy-C4-carnitine by tandem mass spectrometry. (b) Plasma acylcarnitine analysis by tandem mass spectrometry. Left panel: normal acylcarnitine profile; Right panel: acylcarnitine profile from Patient 2 with accumulating hydroxy-C4-carnitine indicated by arrow. Figure 1 HIBCH deficiency leads to accumulation of hydroxy-C4-carnitine. (a) Valine degradation pathway. 3-Hydroxy-isobutyrylCoA hydrolase (HIBCH) catalyses the fifth step of valine catabolism. HIBCH deficiency leads to accumulation of 3-hydroxy-isobutyryl carnitine, which is detected as hydroxy-C4-carnitine by tandem mass spectrometry. (b) Plasma acylcarnitine analysis by tandem mass spectrometry. Left panel: normal acylcarnitine profile; Right panel: acylcarnitine profile from Patient 2 with accumulating hydroxy-C4-carnitine indicated by arrow. The family history is notable in that a maternal uncle of Patients 1 and 2 (whose own parents were first cousins) had a progressive neurological disorder from the age of 14 years, eventually leading to death at 46 years. His sister, the maternal aunt of Patients 1 and 2, had a neuromuscu- lar condition from birth, but normal cognition, and died at 47 years. opmental delay and ataxia and subsequent neurological regression, and his case history has been reported previ- ously [12]. We now present additional biochemical data from this case, including muscle glutathione levels, and compare his neuro-imaging features to those of Patient 2 (Figure 2b). In all three cases, the following diagnostic investigations were performed after informed parental consent. Clinical and biochemical details of these 3 patients, and a fourth patient previously reported in the literature (Brown et al. [11]) are summarised in Table 1. Patient 3 Patient 3 is the first child of healthy unrelated white European parents. He presented at 4 months with devel- iency Patient 2 Patient 3 (previously reported in Loupatty et al. 2007 [12]) Patient reported by Brown et al. 1982 [11] Male Male Male Birth 4 months Birth on Poor feeding Head bobbing Dysmorphic features 2 years 8 months Alive at 8 years 3 months Distantly related parents; younger sibling of Patient 1 Unrelated parents First cousin Egyptian parents Poor feeding Poor feeding Poor feeding ++ + ++ ++ ++ NS hs; 10 months Infantile spasms From 9 months - transient absences and episodes of eye rolling NS + + NS No + NS ep oea, vision hy), hearing Recurrent episodes of screaming, breath-holding, poor sleep, central apnoea, visual im- pairment, microcephaly Cerebellar ataxia - truncal ataxia, dysmetria and intention tremor Facial dysmorphism, tetralogy of Fallot, multiple vertebral anomalies, agenesis of cingulate gyrus and corpus callosum (PM findings) hy in the ncephalo- ised atrophy Abnormal signal within the dentate nuclei and the globi pallidi, with a generalised lack of white matter (Figure 2a) Signal abnormalities in globi pallidi and midbrain and asymmetric involvement of cerebral peduncles (Figure 2b) ND 4.0, 1.6, 1.2 1.7 NS 2.1, 2.6 1.3 NS 0.77-1.25 0.45-1.73 ND se activity) 0.211 0.089 ND 0.056 0.096 ND 0.016 0.013 ND ND 7.9 ND ND 29.5 ND ND ND ~20% of controls us c.219_220insTTGAATAG; are outside the reference http://www.ojrd.com/content/8/1/188 p j Page 6 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Figure 2 Magnetic resonance imaging of the brain in HIBCH deficiency. (a) Patient 2 at 9 months: axial T2 weighted image (left panel) shows bilateral symmetrical signal hyperintensity within the globi pallidi (arrows) accompanied by signal hyperintensity on diffusion weighted image (middle panel) and low signal on ADC map (right panel) in keeping with restricted diffusion which is beginning to pseudonormalise on ADC map. The features are those of a subacute neurometabolic insult. In addition there is some generalised non-specific lack of cerebral volume with prominence of the cerebral sulci and delay in myelin maturation. (b) Patient 3 at 11 months: axial T2 (far left) shows bilateral symmetrical signal hyperintensity and swelling in the globi pallidi (arrows) with restricted diffusion (diffusion weighted image middle panel, ADC map right panel) consistent with cytotoxic oedema. The imaging pattern is suggestive of an acute neurometabolic insult. Figure 2 Magnetic resonance imaging of the brain in HIBCH deficiency. (a) Patient 2 at 9 months: axial T2 weighted image (left panel) shows bilateral symmetrical signal hyperintensity within the globi pallidi (arrows) accompanied by signal hyperintensity on diffusion weighted image (middle panel) and low signal on ADC map (right panel) in keeping with restricted diffusion which is beginning to pseudonormalise on ADC map. The features are those of a subacute neurometabolic insult. In addition there is some generalised non-specific lack of cerebral volume with prominence of the cerebral sulci and delay in myelin maturation. (b) Patient 3 at 11 months: axial T2 (far left) shows bilateral symmetrical signal hyperintensity and swelling in the globi pallidi (arrows) with restricted diffusion (diffusion weighted image middle panel, ADC map right panel) consistent with cytotoxic oedema. The imaging pattern is suggestive of an acute neurometabolic insult. Biochemical analyses electrochemical high performance liquid chromatog- raphy using a previously reported method [17]. Previously reported spectrophotometric assay methods were used to determine the activities of RC enzyme complexes I, II + III and IV in frozen skeletal muscle homogenates [13] and complex IV in cultured skin fi- broblasts [14]. Quantitative immunocytochemistry was used to investigate relative expression of complex IV subunit 1 (MTCO1) in patient and control fibroblasts, which were cultured in the presence of 5 μM MitoTracker® Red CM-H2-XRos (Molecular Probes Inc., Oregon) to label mitochondria red, followed by green immunostaining with an anti-MTCO1 monoclonal antibody (Clone 1D6E 1A8, Abcam) as described [15]. Densitometric analysis of images captured using a Zeiss Axiophot epifluorescence microscope was performed as reported previously [15] and statistical analysis was performed using Student’s t test. PDHc activity was measured in cultured skin fibroblasts using a radiochemical method [16]. HIBCH activity was determined by spectrophotometric enzymatic assay using the physiological substrate S-3-hydroxyiso- butyryl-CoA, as previously described [12]. Glutathione in frozen skeletal muscle homogenates was assayed by Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Discussion Two brothers with a progressive neurodegenerative disorder are reported. Patient 1 had combined mitochon- drial enzyme defects involving PDHc and multiple RC enzymes (complexes I, II + III and IV, Table 1) whereas these were normal in Patient 2. However, the remarkably similar clinical features in both brothers implied that they had the same underlying condition. The presence of persistently elevated plasma hydroxy-C4-carnitine in Patient 2 (Figure 1b, Table 1) suggested the possibility of HIBCH deficiency, which was confirmed by enzyme assay in cultured skin fibroblasts and molecular genetic analysis. Fibroblast HIBCH activity was below detectable limits in both patients (Table 1), and sequence analysis revealed a homozygous missense mutation c.950G>A (p.Gly317Glu) in the HIBCH gene (Figure 4). The causal nature of this mutation is indicated by the fact that this mutation 1) concerns a highly conserved amino acid; 2) is predicted in silico to be deleterious and probably damaging; 3) is the only putative mutation in the coding region of the HIBCH gene encoding HIBCH, an enzyme which was fully deficient in the patients; and 4) has not been observed previously in genome databases, including 200 ethnically matched control alleles. Finally, the Biochemical analyses Patient 1 had combined mitochondrial enzyme defects involving PDHc and multiple RC enzymes: residual enzyme activities in skeletal muscle were 65%, 25% and 71% of the lowest control for complexes I, II + III and IV respectively, whilst fibroblast PDHc activity was 43% of the lowest control (Table 1). RC and PDHc activities were normal in Patient 2, whilst Patient 3 had mild reduction of com- plexes I and IV in skeletal muscle (Table 1). Quantitative immunocytochemistry confirmed significantly decreased expression of complex IV subunit I in cultured skin fibro- blasts from Patient 3 (Figure 3); mean cellular MTCOI expression in Patient 3 fibroblasts was 66% compared to controls (p < 0.001). The presence of persistently elevated plasma hydroxy-C4-carnitine in Patients 2 and 3 suggested the possibility of HIBCH deficiency, which was confirmed by enzyme assay; fibroblast HIBCH activity was below detectable limits in all three patients (Table 1). Molecular analysis of Patient 3 has previously been reported [12]. Results codons downstream). and c.136A>G;p.Thr46Ala. The reported Minor Allele Frequencies for the two polymor- phisms are for c.2T>C: T = 0.3988 (European American) and C = 0.4557 (African American); and for c.136A>G: A = 0.2407 (European American) and A = 0.3443 (African American). Molecular genetic analysis Genomic DNA was extracted from cultured skin fibro- blasts using standard methods. Exons and flanking in- tronic sequences of the HIBCH gene were sequenced after amplification by PCR from genomic DNA using intronic primers with -21 M13 (5'-TGTAAAACGACGGCCAGT-3') or M13-Rev (5'-CAGGAAACAGCTATGACC-3') extensions (Additional file 1: Table S1). PCR fragments were sequenced with -21 M13 and M13-Rev sequence primers using BigDye Terminator cycle sequencing kits (Applied Biosystems, Foster City, CA, USA). Sequence data were compared to the reference HIBCH sequence (GenBank accession num- ber, NM_014362.2) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. All diagnostic investigations were performed after obtaining informed parental consent. This study was approved by the National Research Ethics Committee London Bloomsbury, UK. Page 7 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 codons downstream). and c.136A>G;p.Thr46Ala. The reported Minor Allele Frequencies for the two polymor- phisms are for c.2T>C: T = 0.3988 (European American) and C = 0.4557 (African American); and for c.136A>G: A = 0.2407 (European American) and A = 0.3443 (African American). Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Molecular genetic analysis Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Figure 4 Homozygous HIBCH mutation in patients 1 and 2. Sequence electropherograms of HIBCH gene. Top panel: Control; Second (from top) panel: Patient 1; Third panel: Patient 2; Fourth panel: Mother; Fifth panel: Father. Both patients are homozygous for the c.950G>A mutation, whilst the parents are heterozygous. Figure 4 Homozygous HIBCH mutation in patients 1 and 2. Sequence electropherograms of HIBCH gene. Top panel: Control; Second (from top) panel: Patient 1; Third panel: Patient 2; Fourth panel: Mother; Fifth panel: Father. Both patients are homozygous for the c.950G>A mutation, whilst the parents are heterozygous. Figure 4 Homozygous HIBCH mutation in patients 1 and 2. Sequence electropherograms of HIBCH gene. Top panel: Control; Second (from top) panel: Patient 1; Third panel: Patient 2; Fourth panel: Mother; Fifth panel: Father. Both patients are homozygous for the c.950G>A mutation, whilst the parents are heterozygous. subthalamic regions were also involved in Patient 3. The predilection for the basal ganglia in HIBCH deficiency is intriguing, and the pathomechanism(s) underlying the basal ganglia lesions, and in particular the selective involvement of the globi pallidi and subthalamic nuclei, are not clear. It is possible that the lesions arise from localised cerebral energy failure and subsequent neuronal cell death, as in other mitochondrial diseases including Leigh syndrome (subacute necrotizing encephalomy- elopathy) and Kearns-Sayre syndrome [7,8]. Alternatively, the underlying pathological mechanisms and the imaging changes may more closely resemble those seen in other organic acidurias, such as glutaric aciduria type I and methylmalonic and propionic acidurias, which remain poorly understood [9,10]. However one hypothesis suggests that one mechanism involves reaction of acrylyl-CoA and glutaconyl-CoA with thiol groups [18] and gluta- thione depletion has been noted in methylmalonic acid- aemia (MMA) [19]. It is also interesting to note that there is some evidence for secondary RC defects as a possible mechanism of basal ganglia damage in these organic acidaemias [20,21], and one study demonstrated mutation segregated with the Leigh-like disease in this family and was heterozygous in the unaffected parents and the maternal aunt and uncle who had a different neurological phenotype. Although we performed extensive metabolic investigations in Patient 1 which did not reveal any other metabolic disturbances, we cannot completely exclude the small possibility that a second autosomal recessive disorder may have been present, which could have accounted for the more severe biochemical defects observed in this patient. Molecular genetic analysis Sequence analysis revealed a novel homozygous missense mutation c.950G>A;p.Gly317Glu in the HIBCH gene on chromosome 2q32.3 in both Patients 1 and 2 (Figure 4). The mutation is absent in the dbSNP and 1000 genomes databases (which includes 200 Pakistani alleles), affects an amino acid residue highly conserved among different spe- cies and is predicted by the SIFT software as deleterious and by the Polyphen-2 software as probably damaging. Parents and the maternal aunt and uncle were all het- erozygous for the c.950G>A mutation. Patients 1 and 2 were also homozygous for two known single nucleotide polymorphisms within the HIBCH gene: c.2T>C;p.Met1? (disruption main translation initiation codon; initiation 5 Figure 3 Cytochrome oxidase immunocytochemistry in cultured skin fibroblasts. Expression of complex IV subunit 1 (MTCO1) in control and Patient 3 fibroblasts. Cells were cultured in the presence of 5 μM MitoTracker® Red CM-H2-XRos to label mitochondria red, followed by green immunostaining with anti-MTCO1 antibodies and blue nuclear counterstaining with DAPI. (a) Pseudo-coloured fluorescent micrographs. (b) Relative cellular expression levels of MTCO1 in Control (blue bars) and Patient (red bars) cells. The mean grey levels were measured for both the green and red images of 3631 control and 2184 patient cells, and the ratio of the two signals was calculated for each cell. The histogram reveals the frequency of cells with a particular MTCO1/MitoTracker ratio at intervals of 0.025. The total number of cells per culture is 100%. Figure 3 Cytochrome oxidase immunocytochemistry in cultured skin fibroblasts. Expression of complex IV subunit 1 (MTCO1) in control and Patient 3 fibroblasts. Cells were cultured in the presence of 5 μM MitoTracker® Red CM-H2-XRos to label mitochondria red, followed by green immunostaining with anti-MTCO1 antibodies and blue nuclear counterstaining with DAPI. (a) Pseudo-coloured fluorescent micrographs. (b) Relative cellular expression levels of MTCO1 in Control (blue bars) and Patient (red bars) cells. The mean grey levels were measured for both the green and red images of 3631 control and 2184 patient cells, and the ratio of the two signals was calculated for each cell. The histogram reveals the frequency of cells with a particular MTCO1/MitoTracker ratio at intervals of 0.025. The total number of cells per culture is 100%. Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Page 8 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Ferdinandusse et al. Molecular genetic analysis Increased oxidation of six complex I cysteine residues was demonstrated in glutathione-depleted mouse brains, an oxidative stress model of murine PD [35]. Methacrylyl- CoA acid could further disrupt enzymatic reactions taking place in mitochondria by irreversibly binding cofactors such as CoA and lipoic acid. Lack of CoA would be expected to inhibit the Krebs cycle, whilst lipoate is an essential component of PDHc. competitive inhibition of PDHc by propionyl-CoA, as well as inhibition of complex III and the α-ketoglutarate dehydrogenase complex [22]. Most cases of multiple RC deficiencies reported in the literature are caused by defects of maintenance or translation of the mtDNA. The former group includes defects of nuclear genes involved in replication of the mtDNA and defects of nucleoside salvage necessary to provide dNTP building blocks for mtDNA synthesis [23]. In the previously reported Patient 3, mtDNA copy number was normal in skeletal muscle (Table 1) and cultured myoblasts and skin fibroblasts (data not shown), excluding the possibility of mtDNA depletion as the cause of the mild RC defects observed in this patient. Disorders of mitochondrial translation may be primary mtDNA defects, such as deletions of mtDNA or point mutations involving the mitochondrial transfer RNA genes, or nuclear gene defects [3]. In a small number of cases, a combined deficiency of multiple RC enzymes associated with PDHc deficiency has been reported, the so-called multiple mitochondrial dysfunctions syndrome. Recently, mutations in two Fe-S cluster assembly proteins were shown to cause multiple mitochondrial enzyme defects including PDHc deficiency. Mutations in NFU1, required late in the maturation of Fe-S clusters, caused a syndrome of fatal infantile encephalopathy and/or pulmonary hyper- tension in Spanish and Mexican families with combined defects of PDHc, complex II, lipoic acid synthase and the glycine cleavage system [5,6]. Patients with mutations in BOLA3, which is also needed for Fe-S and lipoic acid biosynthesis, have a similar biochemical phenotype [6,24], but the precise pattern of RC deficiencies in these defects differs according to the specific Fe-S cluster synthesis pathway that is impaired [6]. y With respect to the pathological mechanism under- lying HIBCH deficiency, methacrylyl-CoA, a proximal metabolite in the valine degradation pathway (Figure 1a), could well play a causative role. Cysteine and cysteamine conjugates of methacrylyl-CoA were shown to accumulate in multiple tissues, particularly liver, kidney and brain, in the first patient reported with HIBCH deficiency [11]. Molecular genetic analysis HIBCH deficiency has been reported in only two cases previously including Patient 3 [11,12]. Table 1 summa- rises the clinical and biochemical findings in all 4 cases. Consistent features of this condition appear to be hypotonia, poor feeding and developmental regression with seizures starting in infancy (Table 1) and symmetrical involvement of the globi pallidi seen on brain MRI. Images were no longer available from Patient 1 for review, but acute changes suggestive of cytotoxic oedema, shown by imaging changes of restricted diffusion with subsequent evolution to mature scarring, were seen in Patient 2 and also in Patient 3 (previously reported in [12]). The Page 9 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 aconitase), since cysteine provides sulphide for Fe-S clusters. The ability of thiol groups to undergo redox reactions confers antioxidant properties to cysteine, most importantly in the tripeptide glutathione (composed of cysteine, glycine and glutamic acid) and also in mitochon- drial thioredoxin. Thioredoxin contains two cysteine residues in its active centre that undergo reversible oxidation/reduction in response to mitochondrial redox status [30]. Depletion of mitochondrial pools of cysteine, glutathione and thioredoxin by methacrylyl-CoA could lead to oxidative damage, with further impairment of RC enzyme function. Indeed, we found mildly reduced muscle glutathione in Patient 3, who also had mildly reduced activities of RC complexes I and IV in muscle (Table 1), and significantly decreased expression of complex IV subunit I in fibroblasts measured by quantitative im- munocytochemistry (Figure 3). These effects are likely to vary according to the levels of oxidative stress, which may explain the variable RC and PDHc defects seen in the 3 patients with HIBCH deficiency (Table 1). We have previously demonstrated reversibility of RC defects in Patient 3, who had normal activities of complexes I-IV in a biopsy performed 3 months after the abnormal biopsy shown in Table 1 with mildly reduced complex I and IV levels (Loupatty et al. 2007 [12]). A reduced plasma concentration of glutathione has been found in both mitochondrial disorders and organic acidaemias [31,32]. Complex I dysfunction was linked to Parkinson’s disease (PD) following the observation of a high incidence of PD in subjects who had recreationally misused 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a known inhibitor of complex I [33] and glutathione depletion has been noted to precede complex I deficiency in PD [34]. Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Molecular genetic analysis Cysteine is a non-essential amino acid bearing a thiol side chain that is frequently involved in enzymatic reactions and is susceptible to oxidation, leading to formation of the disulphide cystine. Cystine plays an important structural role in many proteins; disulphide bonds are important in crosslinking proteins, serving to increase rigidity and conferring resistance to proteolytic degradation. Methacry- lyl-CoA could react with mitochondrial enzymes containing essential cysteine residues, including PDHc and RC enzymes [25-29], thereby reducing their activities. De- pletion of cysteine also could lead to reduced activity of mitochondrial enzymes containing Fe-S clusters (e.g. RC complexes I, II and III and the Krebs cycle enzyme Toxic damage to mitochondrial enzymes has previously been demonstrated in ethylmalonic encephalopathy. In affected patients mutations of the ETHE1 sulphur diox- ygenase lead to accumulation of sulphide, which affects the function of complex IV and the short chain acyl-CoA dehydrogenase involved in mitochondrial fatty acid oxida- tion [36,37]. By contrast, complex IV activity appears to be only mildly reduced in HIBCH deficiency, with rela- tively greater effects on complex I and PDHc observed in Patient 1 in this study. These cases illustrate the clinical utility of acylcarnitine analysis in the differential diagnosis of suspected mito- chondrial disease. The older brother Patient 1 had died Page 10 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 Page 10 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 before the introduction of blood acylcarnitine analysis into routine metabolic clinical practice, but the persistently elevated hydroxy-C4-carnitine levels in his younger brother Patient 2 eventually led to the correct diagnosis of HIBCH deficiency. Acylcarnitine analysis is a useful investigation in other cases of suspected mitochondrial disease, since it may help to direct diagnostic genetic investigations, for example succinyl and propionyl carnitines may be elevated in patients with succinyl-CoA ligase deficiency caused by SUCLA2 or SUCLG1 mutations [38,39]. 3. Chrzanowska-Lightowlers ZM, Horvath R, Lightowlers RN: 175th ENMC International Workshop: mitochondrial protein synthesis in health and disease, 25-27th June 2010, Naarden, The Netherlands. Neuromuscul Disord 2011, 21:142–147. 4. Rouault TA: Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease. Dis Model Mech 2012, 5:155–164. 5. Conclusions 7. Rahman S, Blok RB, Dahl HH, Danks DM, Kirby DM, Chow CW, Christodoulou J, Thorburn DR: Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 1996, 39:343–351. 7. Rahman S, Blok RB, Dahl HH, Danks DM, Kirby DM, Chow CW, Christodoulou J, Thorburn DR: Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 1996, 39:343–351. In conclusion, HIBCH deficiency should be considered in the differential diagnosis for patients with multiple RC defects, including those with associated PDHc deficiency. Moreover, the presence of hydroxy-C4-carnitine in the acylcarnitine profile should alert the clinician to the possi- bility of this condition. Our results stress the importance of acylcarnitine analysis in patients with suspected mito- chondrial disease. 8. Valanne L, Ketonen L, Majander A, Suomalainen A, Pihko H: Neuroradiologic findings in children with mitochondrial disorders. AJNR Am J Neuroradiol 1998, 19:369–377. 8. Valanne L, Ketonen L, Majander A, Suomalainen A, Pihko H: Neuroradiologic findings in children with mitochondrial disord AJNR Am J Neuroradiol 1998, 19:369–377. 9. Harting I, Neumaier-Probst E, Seitz A, Maier EM, Assmann B, Baric I, Troncoso M, Muhlhausen C, Zschocke J, Boy NP, et al: Dynamic changes of striatal and extrastriatal abnormalities in glutaric aciduria type I. Brain 2009, 132:1764–1782. 10. Harting I, Seitz A, Geb S, Zwickler T, Porto L, Lindner M, Kolker S, Horster F: Looking beyond the basal ganglia: the spectrum of MRI changes in methylmalonic acidaemia. J Inherit Metab Dis 2008, 31:368–378. Molecular genetic analysis Navarro-Sastre A, Tort F, Stehling O, Uzarska MA, Arranz JA, Del TM, Labayru MT, Landa J, Font A, Garcia-Villoria J, et al: A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins. Am J Hum Genet 2011, 89:656–667. 6. Cameron JM, Janer A, Levandovskiy V, Mackay N, Rouault TA, Tong WH, Ogilvie I, Shoubridge EA, Robinson BH: Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes. Am J Hum Genet 2011, 89:486–495. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 13. Hargreaves P, Rahman S, Guthrie P, Taanman JW, Leonard JV, Land JM, Heales SJ: Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion. J Inherit Metab Dis 2002, 25:7–16. Authors’ contributions SF performed biochemical studies, interpreted data and revised manuscript. HRW performed molecular genetic studies, interpreted data and revised manuscript. SJRH, GB, IPH, J-WT, RG, LA, RJAW acquired and interpreted data. PTC and JVL interpreted data and revised manuscript. SR conceived study, acquired and interpreted data, and wrote manuscript. All authors read and approved the final manuscript. 14. Hayasaka K, Brown GK, Danks DM, Droste M, Kadenbach B: Cytochrome c oxidase deficiency in subacute necrotizing encephalopathy (Leigh syndrome). J Inherit Metab Dis 1989, 12:247–256. 15. Taanman JW, Muddle JR, Muntau AC: Mitochondrial DNA depletion can be prevented by dGMP and dAMP supplementation in a resting culture of deoxyguanosine kinase-deficient fibroblasts. Hum Mol Genet 2003, 12:1839–1845. Author details 1 1Academic Medical Centre, Laboratory Genetic Metabolic Diseases, University of Amsterdam, Amsterdam, The Netherlands. 2Chemical Pathology, Great Ormond Street Hospital, London, UK. 3Diagnostic Imaging, Great Ormond Street Hospital, London, UK. 4Metabolic Unit, Great Ormond Street Hospital, London, UK. 5Neurometabolic Unit, National Hospital for Neurology, London, UK. 6Mitochondrial Research Group, Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. 7Department of Biochemistry, University of Oxford, Oxford, UK. 8Department of Clinical Neurosciences, UCL Institute of Neurology, London, UK. 17. Hargreaves IP, Sheena Y, Land JM, Heales SJ: Glutathione deficiency in patients with mitochondrial disease: implications for pathogenesis and treatment. J Inherit Metab Dis 2005, 28:81–88. 18. Lehnert W, Sass JO: Glutaconyl-CoA is the main toxic agent in glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I). Med Hypotheses 2005, 65:330–333. 19. Treacy E, Arbour L, Chessex P, Graham G, Kasprzak L, Casey K, Bell L, Mamer O, Scriver CR: Glutathione deficiency as a complication of methylmalonic acidemia: response to high doses of ascorbate. J Pediatr 1996, 129:445–448. 20. Okun JG, Horster F, Farkas LM, Feyh P, Hinz A, Sauer S, Hoffmann GF, Unsicker K, Mayatepek E, Kolker S: Neurodegeneration in methylmalonic aciduria involves inhibition of complex II and the tricarboxylic acid cycle, and synergistically acting excitotoxicity. J Biol Chem 2002, 277:14674–14680. Received: 22 June 2013 Accepted: 9 November 2013 Published: 4 December 2013 Additional file 11. Brown GK, Hunt SM, Scholem R, Fowler K, Grimes A, Mercer JF, Truscott RM, Cotton RG, Rogers JG, Danks DM: Beta-hydroxyisobutyryl coenzyme a deacylase deficiency: a defect in valine metabolism associated with physical malformations. Pediatrics 1982, 70:532–538. Additional file 1: Table S1. HIBCH-specific PCR primers used for sequence analysis of HIBCH. 12. Loupatty FJ, Clayton PT, Ruiter JP, Ofman R, Ijlst L, Brown GK, Thorburn DR, Harris RA, Duran M, Desousa C, et al: Mutations in the gene encoding 3- hydroxyisobutyryl-CoA hydrolase results in progressive infantile neurodegeneration. Am J Hum Genet 2007, 80:195–199. Acknowledgements g SR and PTC are supported by Great Ormond Street Hospital Children’s Charity. SR and PTC are supported by Great Ormond Street Hospital Children’s Charity. 16. Wicking CA, Scholem RD, Hunt SM, Brown GK: Immunochemical analysis of normal and mutant forms of human pyruvate dehydrogenase. Biochem J 1986, 239:89–96. 2. Sarzi E, Bourdon A, Chretien D, Zarhrate M, Corcos J, Slama A, de Lonlay P, Cormier-Daire V, Rotig A, Munnich A: Mitochondrial DNA depletion is a prevalent cause of multiple respiratory chain deficiency in childhood. J Pediatr 2007, 150:531–534. 1. Rahman S, Hanna MG: Diagnosis and therapy in neuromuscular disorders: diagnosis and new treatments in mitochondrial diseases. J Neurol Neurosurg Psychiatry 2009, 80:943–953. References 21. de Keyzer Y, Valayannopoulos V, Benoist JF, Batteux F, Lacaille F, Hubert L, Chretien D, Chadefeaux-Vekemans B, Niaudet P, Touati G, et al: Multiple OXPHOS deficiency in the liver, kidney, heart, and skeletal muscle of patients with methylmalonic aciduria and propionic aciduria. Pediatr Res 2009, 66:91–95. References 1. Rahman S, Hanna MG: Diagnosis and therapy in neuromuscular disorders: diagnosis and new treatments in mitochondrial diseases. J Neurol Neurosurg Psychiatry 2009, 80:943–953. 2. Sarzi E, Bourdon A, Chretien D, Zarhrate M, Corcos J, Slama A, de Lonlay P, Cormier-Daire V, Rotig A, Munnich A: Mitochondrial DNA depletion is a prevalent cause of multiple respiratory chain deficiency in childhood. J Pediatr 2007, 150:531–534. 1. Rahman S, Hanna MG: Diagnosis and therapy in neuromuscular disorders: diagnosis and new treatments in mitochondrial diseases. J Neurol Neurosurg Psychiatry 2009, 80:943–953. 22. Schwab MA, Sauer SW, Okun JG, Nijtmans LG, Rodenburg RJ, van den Heuvel LP, Drose S, Brandt U, Hoffmann GF, Ter LH, et al: Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins. Biochem J 2006, 398:107–112. Page 11 of 11 Page 11 of 11 Ferdinandusse et al. Orphanet Journal of Rare Diseases 2013, 8:188 http://www.ojrd.com/content/8/1/188 23. Rahman S, Poulton J: Diagnosis of mitochondrial DNA depletion syndromes. Arch Dis Child 2009, 94:3–5. 24. Haack TB, Rolinski B, Haberberger B, Zimmermann F, Schum J, Strecker V, Graf E, Athing U, Hoppen T, Wittig I, et al: Homozygous missense mutation in BOLA3 causes multiple mitochondrial dysfunctions syndrome in two siblings. J Inherit Metab Dis 2012. 25. Schwartz ER, Reed LJ: Alpha-keto acid dehydrogenase complexes. 13. Reaction of sulfhydryl groups in pyruvate dehydrogenase with organic mercurials. J Biol Chem 1970, 245:183–187. 26. Khailova LS, Korochkina LG, Severin SE: Organization and functioning of muscle pyruvate dehydrogenase active centers. Ann N Y Acad Sci 1989, 573:36–54. 27. Ali MS, Roche TE, Patel MS: Identification of the essential cysteine residue in the active site of bovine pyruvate dehydrogenase. J Biol Chem 1993, 268:22353–22356. 28. Burwell LS, Nadtochiy SM, Tompkins AJ, Young S, Brookes PS: Direct evidence for S-nitrosation of mitochondrial complex I. Biochem J 2006, 394:627–634. 29. Cooperstein SJ: Reversible inactivation of cytochrome oxidase by disulfide bond reagents. J Biol Chem 1963, 238:3606–3610. y disulfide bond reagents. J Biol Chem 1963, 238:3606–3610. 30. Ferdinandusse et al. 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Tiranti V, Viscomi C, Hildebrandt T, Di MI, Mineri R, Tiveron C, Levitt MD, Prelle A, Fagiolari G, Rimoldi M, et al: Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy. Nat Med 2009, 15:200–205. 38. Elpeleg O, Miller C, Hershkovitz E, Bitner-Glindzicz M, Bondi-Rubinstein G, Rahman S, Pagnamenta A, Eshhar S, Saada A: Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion. Am J Hum Genet 2005, 76:1081–1086. 39. Van Hove JL, Saenz MS, Thomas JA, Gallagher RC, Lovell MA, Fenton LZ, Shanske S, Myers SM, Wanders RJ, Ruiter J, et al: Succinyl-CoA ligase deficiency: a mitochondrial hepatoencephalomyopathy. Pediatr Res 2010, 68:159–164. doi:10.1186/1750-1172-8-188 Cite this article as: Ferdinandusse et al.: HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase. Orphanet Journal of Rare Diseases 2013 8:188. References doi:10.1186/1750-1172-8-188 Cite this article as: Ferdinandusse et al.: HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase. Orphanet Journal of Rare Diseases 2013 8:188. 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/W4254338219	https://www.qeios.com/read/0AFQCQ/pdf	English	null	Migrating Partial Seizures in Infancy	Definitions	2,020	cc-by	98	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Migrating Partial Seizures in Infancy National Cancer Institute National Cancer Institute Qeios ID: 0AFQCQ · https://doi.org/10.32388/0AFQCQ Source National Cancer Institute. Migrating Partial Seizures in Infancy. NCI Thesaurus. Code C125387. C125387. A very rare severe form of epilepsy with poor prognosis that usually begins within a few weeks of birth. The seizure activity can appear in multiple locations in the brain or migrate from one region to another during an episode. It results in severe developmental delay. Qeios ID: 0AFQCQ · https://doi.org/10.32388/0AFQCQ 1/1
https://openalex.org/W4387898380	https://www.frontiersin.org/articles/10.3389/fsurg.2023.1274313/pdf?isPublishedV2=False	English	null	Therapeutic evaluation and analysis of complications of ethanol sclerotherapy for intramuscular vascular malformations: a single-center retrospective study	Frontiers in surgery	2,023	cc-by	5,864	Therapeutic evaluation and analysis of complications of ethanol sclerotherapy for intramuscular vascular malformations: a single-center retrospective study Cai-Jun Jin 1†, Qian Wang 2†, Min Wang 2, Yong Chen 2 and Si-Ming Yuan 1,2* Cai-Jun Jin 1†, Qian Wang 2†, Min Wang 2, Yong Chen 2 and Si-Ming Yuan 1,2* †These authors have contributed equally to this work 1Department of Plastic Surgery, Jinling Hospital, Nanjing Medical University, Nanjing, China, 2Department of Plastic Surgery, Jinling Hospital, Southern Medical University, Nanjing, China RECEIVED 08 August 2023 ACCEPTED 19 September 2023 PUBLISHED 24 October 2023 CITATION Jin C-J, Wang Q, Wang M, Chen Y and Yuan S-M (2023) Therapeutic evaluation and analysis of complications of ethanol sclerotherapy for intramuscular vascular malformations: a single-center retrospective study. Background: Intramuscular venous malformations (IMVMs) can cause pain and contracture deformity, leading to dysfunction of limbs. Ethanol sclerotherapy is one of the main treatments for IMVMs. This study aims to evaluate the efﬁcacy and the complications associated with intravascular ethanol sclerotherapy for IMVMs and to provide a comprehensive summary of clinical experiences for future reference. CITATION Jin C-J, Wang Q, Wang M, Chen Y and Yuan S-M (2023) Therapeutic evaluation and analysis of complications of ethanol sclerotherapy for intramuscular vascular malformations: a single-center retrospective study. Front. Surg. 10:1274313. Methods: A retrospective analysis was conducted on a cohort of 118 patients diagnosed with IMVMs who were treated with ethanol sclerotherapy in our center between 2006 and 2021. The plastic surgeons utilized a standardized collection pro forma to record the clinical data. Furthermore, a follow-up period ranging from 6 months to 5 years was implemented to assess the relief of symptoms, the change of lesion size, and the recovery of functional outcomes. In addition, an analysis of long-term complications was conducted. doi: 10.3389/fsurg.2023.1274313 COPYRIGHT © 2023 Jin, Wang, Wang, Chen and Yuan. 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. Results: The clinical symptoms of the patients in this group included pain, swelling, and limited movement. On average, 5.61 mL (range 2–14 mL) of ethanol was used during the sclerotherapy procedure. TYPE Original Research PUBLISHED 24 October 2023 DOI 10.3389/fsurg.2023.1274313 intramuscular venous malformations, ethanol, sclerotherapy, safety, efﬁcacy Therapeutic evaluation and analysis of complications of ethanol sclerotherapy for intramuscular vascular malformations: a single-center retrospective study The intraoperative and early postoperative complications were successfully relieved by means of timely intervention, as observed during the follow-up period. Based on the MRI results, the sizes of the lesions in 19% of the cases were signiﬁcantly decreased, while a slight decrease was observed in 39% of the cases. During the follow-up period, it was found that only eight out of the 118 patients included in this study experienced long- term complications related to sclerotherapy. Conclusions: Although ethanol sclerotherapy has proven to be an effective ﬁrst- line treatment for IMVMs, it is associated with a variety of adverse reactions and short- and long-term complications. Surgeons are required to perform operations prudently and provide timely medical intervention for postoperative complications. intramuscular venous malformations, ethanol, sclerotherapy, safety, efﬁcacy Frontiers in Surgery 01 frontiersin.org 10.3389/fsurg.2023.1274313 10.3389/fsurg.2023.1274313 Jin et al. Complication analysis After a sclerotherapy treatment, vital signs and blood supply to the skin around the injection point were closely monitored, and complications were recorded, such as blood supply disorder, ulceration, infection, nerve injury, superﬁcial vein thrombosis, muscle contracture, and joint deformity. Study design and patients This study was reviewed and approved by the Medical Ethics Committee of Jinling Hospital. The study complies with the Declaration of Helsinki. Informed consent for the treatment and the publication of images was obtained from all the patients. The participants included in this study were individuals diagnosed with IMVMs who were treated with ethanol sclerotherapy in our center between January 2006 and December 2021. The sample consisted of 77 female and 41 male patients, aged 4–59 years with an average age of 23.17. At admission, all the patients underwent an MRI examination. For patients with several treatments, an MRI examination was performed after three consecutive treatment sessions. The patients who received fewer than three treatment sessions would be scheduled for an MRI examination prior to their last follow-up. The changes in the size of the lesion were evaluated by measuring the maximum diameter observed using MRI before and after treatment with the following categorization: (1) no signiﬁcant changes if less than 10%; (2) moderate reduction between 10% and 50%; and (3) marked reduction if higher than 50%. The inclusion criteria are as follows: (1) patients diagnosed with IMVM by MRI; (2) patients with signs or symptoms; (3) patients who had undergone ethanol sclerotherapy; and (4) patients with pre- and post-treatment data, followed up for more than 6 months. The exclusion criteria are as follows: (1) digital subtraction angiography or MRI revealing arteriovenous malformations, capillary malformations, lymphatic malformations, or combined vascular malformations; (2) patients who received foam sclerotherapy, intralesional copper wire retention or surgical resection and other treatments; and (3) patients with incomplete clinical data. Introduction intravenous anesthesia or local inﬁltration anesthesia was administered according to the pain tolerance of the patients. Prior to the sclerotherapy procedure, all patients were administered 0.3 mg/kg of methylprednisolone intravenously to reduce the risk of complications such as cough and chest tightness. Based on the ﬁndings of the preoperative MRI and further imaging examinations, the lesion’s precise location was identiﬁed and subsequently accessed through percutaneous puncture using a 22-gauge scalp needle attached to a 10-mL syringe. The accuracy of needle positioning in the venous pouch was ascertained based on the venous blood reﬂux without obvious arterial pulsation through the needle. Some small lesions were punctured under ultrasound assistance. The treatment consisted of multifocal and repeated injections. The total amount of ethanol used in each treatment did not exceed 0.2 mL/kg. Throughout the injection process, vital signs and blood supply to the skin around the injection point were closely monitored. The treatment procedure was repeated after 2–3 months. Following the third treatment, an MRI scan was requested to evaluate the therapeutic effectiveness of the intervention and inform the determination of the subsequent treatment plan. As the most common congenital vascular anomalies, venous malformations (VMs) could occur in many tissues or organs, including the skin and muscles, with approximately 40% of cases occurring in skeletal muscles. At birth, patients with intramuscular venous malformations (IMVMs) typically exhibit few symptoms and indications. As the patients grow, or encounter trauma, puberty, pregnancy, or other factors, the lesion can progressively enlarge, resulting in worsening of symptoms, such as pain, swelling, and even motor limitation. Therefore, timely treatment is needed for IMVM patients to alleviate the symptoms and control the size of the lesions. At present, there are various therapies for the management of IMVMs, including conservative treatment, sclerotherapy, operations, laser treatment, and cryoablation, among which sclerotherapy is the most commonly used. There are also many kinds of sclerosants available. Ethanol is the most widely used sclerosant in our center due to its high efﬁcacy and potent destructive properties. Meanwhile, some scholars expressed that future studies should be directed toward investigating the anatomic location and the depth of the malformations. Unfortunately, there is a lack of systematic research pertaining to the utilization of ethanol sclerotherapy for IMVM. In this study, a retrospective analysis was conducted to evaluate the therapeutic effectiveness and complications associated with ethanol sclerotherapy in IMVMs. Frontiers in Surgery Outcome assessment and follow-up The patients in this group were followed up for a period of 6 months to 5 years, with an average of 41 months. We followed up with patients at the outpatient clinic. Those who could not be followed up through personal visits at the clinic were evaluated by a validated phone questionnaire. The details of the follow-up included the therapeutic effects of absolute ethanol sclerotherapy on relieving clinical symptoms such as pain and swelling. To assess the outcome in a more scientiﬁc and objective manner, the degree of symptom relief was divided into three categories: improvement, no change, and deterioration. frontiersin.org Treatment Following admission, all patients underwent routine examination to exclude surgery contraindications. General Complications were categorized based on the timing of their occurrence: (1) intraoperative complications: complications that Frontiers in Surgery 02 frontiersin.org frontiersin.org Jin et al. 10.3389/fsurg.2023.1274313 occurred immediately during the operation, including cough, chest tightness, and allergic reactions; (2) early postoperative complications: occurring within 1 month after sclerotherapy but could be cured by conservative treatment, including ulceration, infection, superﬁcial vein embolization, and hematuria; (3) late postoperative complications: occurring beyond 1 month after sclerotherapy, including muscle contracture, joint deformity, and peripheral nerve injury. TABLE 1 Characteristics of patients. N Percent (%) Average Range Sex Male 41 35 Female 77 65 Age of presentation 28.53 12–56 Age of onset 18.84 11–42 Length of stay 1.89 1–5 Location of IMVM Calf 27 23 Thigh 32 27 Upper extremities 22 19 Trunk 8 7 Head and neck 11 9 Foot 18 15 Symptoms Pain 109 92 Swelling 62 53 Motion limitation 39 33 Morphology of lesions Focal mass type 64 54 Focal inﬁltration type 32 27 Diffuse inﬁltration type 22 19 Number of lesions 1 77 65 2 25 21 >2 16 14 Sclerotherapy treatment The cohort of 118 patients underwent a total of 378 treatment sessions (1–10 times per patient), with an average of 3.20 sessions per patient. In each session, 2–16 mL of absolute ethanol was injected, with an average of 5.61 mL and a maximum dosage of 0.2 mL/kg per patient. All patients were routinely examined using MRI at the ﬁrst admission to evaluate the range and location of the lesions. Based on the imaging ﬁndings, IMVM was categorized into three types: (1) Focal mass type (n = 64): the lesions were localized with well-deﬁned margins. (2) Focal inﬁltration type (n = 32): the lesions were diffused in one muscle. (3) Diffuse inﬁltration type (n = 22): the lesions were extensive in more than one muscle with margins ill-deﬁned. Patient characteristics A total of 118 patients with IMVM were enrolled in this study. IMVM lesions can be located all over the body, such as in the lower leg (n = 27), thigh (n = 32), upper limb (n = 22), trunk (n = 8), face (n = 11), and foot (n = 18). Meanwhile, 90 patients experienced pain, with 19 patients exhibiting symptoms of persistent pain, 39 patients reporting limited movement, and 62 patients presenting with swelling. The relevant demographic data, lesion location, symptoms, and imaging ﬁndings are shown in Table 1. Statistical analysis Statistical analysis was performed using IBM’s SPSS Statistics 25.0 for Windows. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation (X ± SD), and a t-test was performed for comparisons between the two groups. Fisher’s exact test was used for categorical data. Multiple groups were compared using the Kruskal–Wallis rank- sum test. A P-value < 0.05 was considered to indicate a statistically signiﬁcant difference. Complication analysis challenging to entirely eliminate the lesions with surgical intervention. In addition, it may lead to serious postoperative complications such as bleeding and muscle and nerve injury. Sclerotherapy is the most commonly used treatment for IMVM. This treatment involves injecting sclerosing agents or chemicals into the malformed veins, contacting with the vascular endothelium, which will result in aseptic inﬂammation, thrombosis, and ﬁbrosis (3). Compared with surgical resection, sclerotherapy alleviates the burden and shortens hospital stay. Therefore, sclerotherapy should be preferred for IMVM patients with focal or diffuse lesions. The incidence of complications related to sclerotherapy is shown in Table 3, including the following: (1) intraoperative complications: cough (47/118) and chest tightness (13/118); (2) early postoperative complications: skin ulceration (15/118), superﬁcial vein embolization (2/118), hematuria (39/118), and infection (3/118); and (3) late postoperative complications: muscle contracture (5/118), joint deformity (2/118), and peripheral nerve injury (3/118). The above complications were relieved after symptomatic treatment. Life- threatening complications such as hemolysis, heart failure, pulmonary embolism, and allergic reactions were not observed. A wide variety of sclerosants are available, such as absolute ethanol, monoethanolamine oleate, bleomycin, sodium tetradecyl sulfate (STS), and polidocanol (4). According to the form, they can be also classiﬁed into liquid sclerosants and foam sclerosants. Ethanol is considered to be the most effective and destructive liquid sclerosant for treating lesions (5). In contrast, foam sclerosants such as STS and polidocanol are less destructive for tissues and thus needs more treatment sessions. In this study, all the subjects were treated using ethanol. Out of all the patients studied, 109 patients experienced pain, of which 63 (58%) patients demonstrated improvement; 62 patients experienced swelling, of which 34 (55%) patients had improved; and 39 patients had combined limitation of motion, of which 17 (44%) patients had improved. Frontiers in Surgery Outcome assessment and follow-up bThe distribution of grade information in the “pain” group is statistically different, H = 13.130, P = 0.001. cThe data in this group are statistically different from that in the other two groups. Outcome assessment and follow-up The follow-up data of 118 patients are shown in Table 2. Among the 118 patients, 109 experienced pain, of which 63 (58%) cases had improved after ethanol sclerotherapy. A total of 62 patients were complicated with swelling, of which 34 (55%) cases had improved. A total of 39 patients experienced motion limitation, of which 17 (44%) cases had improved. Among them, there was a statistically signiﬁcant difference in the improvement of pain among the three groups (H = 13.130, P = 0.001). In addition, there was a statistically signiﬁcant difference in the swelling of patients not only between focal mass type and focal inﬁltration type (P = 0.015) but also between focal mass type and diffuse inﬁltration type (P = 0.009). Under MRI examinations, the lesions of 22 patients (19%) showed a remarkable reduction in size, as the typical cases shown in Figures 1–3. The lesions of 46 patients (39%) showed a moderate reduction in size, while that of 50 patients (42%) showed no signiﬁcant change. There were no statistically signiﬁcant differences observed in the levels of pain experienced by patients in each group. However, there was a statistically signiﬁcant difference in swellings of patients not only between focal mass type and focal inﬁltration type (P = 0.001) but also between focal mass type and diffuse inﬁltration type (P ≈0.000), while there was no statistically signiﬁcant difference observed between focal inﬁltration type and diffuse inﬁltration type (P > 0.05). The difference in motion limitation was statistically signiﬁcant between not only focal mass type and focal inﬁltration type (P ≈0.000) but also focal mass type diffuse inﬁltration type (P = 0.000). Still, there was no statistically signiﬁcant difference between focal inﬁltration type and diffuse inﬁltration type (P > 0.05). 03 Frontiers in Surgery frontiersin.org Jin et al. 10.3389/fsurg.2023.1274313 TABLE 2 Ethanol sclerotherapy and its outcomes. TABLE 2 Ethanol sclerotherapy and its outcomes. frontiersin.org Outcome assessment and follow-up Focal mass type (n = 64) Focal inﬁltration type (n = 32) Diffuse inﬁltration type (n = 22) All Overall sessions 222 86 70 378 Sclerosing sessions per patients 3.47 2.69 3.18 3.2 (1–10)a Does of sclerosants 4.68 (2–8)a 5.97 (4–12)a 7.54 (6–14)a 5.61 (2–14)a Clinical results Painb (n = 109) 61 28 20 109 Improved 45 11 7 63 No change 14 17 12 43 Worsened 2 0 1 3 Swelling (n = 62) 22c 22 18 62 Improved 10 13 11 34 No change 12 9 7 28 Worsened 0 0 0 0 Motion limitation (n = 39) 7c 17 15 39 Improved 4 8 5 17 No change 2 8 8 18 Worsened 1 1 2 4 MRI results Marked reduction 14 4 4 22 Moderate reduction 26 13 7 46 No signiﬁcant change 24 15 11 50 aThe average (minimum–maximum). bThe distribution of grade information in the “pain” group is statistically different, H = 13.130, P = 0.001. cThe data in this group are statistically different from that in the other two groups. Focal mass type (n = 64) Focal inﬁltration type (n = 32) Diffuse inﬁltration type (n = 22) All Overall sessions 222 86 70 378 Sclerosing sessions per patients 3.47 2.69 3.18 3.2 (1–10)a Does of sclerosants 4.68 (2–8)a 5.97 (4–12)a 7.54 (6–14)a 5.61 (2–14)a Clinical results Painb (n = 109) 61 28 20 109 Improved 45 11 7 63 No change 14 17 12 43 Worsened 2 0 1 3 Swelling (n = 62) 22c 22 18 62 Improved 10 13 11 34 No change 12 9 7 28 Worsened 0 0 0 0 Motion limitation (n = 39) 7c 17 15 39 Improved 4 8 5 17 No change 2 8 8 18 Worsened 1 1 2 4 MRI results Marked reduction 14 4 4 22 Moderate reduction 26 13 7 46 No signiﬁcant change 24 15 11 50 aThe average (minimum–maximum). bThe distribution of grade information in the “pain” group is statistically different, H = 13.130, P = 0.001. cThe data in this group are statistically different from that in the other two groups. g ( ) bThe distribution of grade information in the “pain” group is statistically different, H = 13.130, P = 0.001. cThe data in this group are statistically different from that in the other two groups. Discussion IMVM is a congenital chronic refractory disease, and the lesions are mostly located in the muscles of limbs, showing progressive growth (1). Most patients experienced varying degrees of pain, swelling, or motion limitation. More critically, the presence of limb contracture deformity can signiﬁcantly decline the patients’ quality of life. Although VM is a benign lesion, it has the characteristics of destructibility and invasiveness, leading to the lack of muscle coherence, reduced muscle contractility, and potential bone deformities. With regard to complications following ethanol sclerotherapy, some researchers have conﬁrmed that 0.25 mL/kg of ethanol is considered to be the critical threshold that may cause adverse reactions (6). In addition, the locations of IMVM lesions are relatively deep, hence reducing the likelihood of skin necrosis following ethanol sclerotherapy (7). The injection dose was Currently, the treatment of IMVM is controversial. Some scholars believe that surgical resection should be the primary therapy for focal mass type, by which the deformed venous lesions can be completely removed (2). However, due to IMVMs invasive nature and deep location within the muscles, it is 04 frontiersin.org Jin et al. 10.3389/fsurg.2023.1274313 FIGURE 1 Focal mass type IMVM in the right neck. (A) The appearance before treatment. (B) The MRI before treatment. (C) The appearance after two times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgure were held by corresponding author. FIGURE 1 Focal mass type IMVM in the right neck. (A) The appearance before treatment. (B) The MRI before treatment. (C) The appearance after two times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgure were held by corresponding author. which reﬂuxed into the pulmonary capillaries and triggered choking. However, it was relieved by preoperative administration of hydrogenated prednisone and a brief period of intraoperative rest. controlled to less than 0.2 mL/kg. Patients were required for follow- up visits every 3 months, and the subsequent treatments were mainly based on symptom improvement or imaging results. The appropriate dose of ethanol for each injection and the multiple injections at an interval of 3 months can effectively reduce the occurrence of complications and achieve optimal efﬁcacy step by step. The common complications of ethanol sclerotherapy include intraoperative complaints of chest tightness and dyspnea. However, administering glucocorticoids prior to treatment can effectively relieve these symptoms (8). Frontiers in Surgery frontiersin.org Discussion The complications were categorized based on the timing of their occurrence into three groups: intraoperative complications, early postoperative complications, and late postoperative complications. In our study, intraoperative complications occurred in 51 patients, including 47 cases of cough and 13 cases of chest tightness, of which nine patients experienced both cough and chest tightness. No allergic reactions were observed. The above patients probably developed microthrombi in the blood vessels during ethanol sclerotherapy, Petechiae and local skin necrosis observed in 15 patients were among the early postoperative complications in this study, of which 13 patients recovered completely after symptomatic treatment manifesting as improved microcirculation and enhanced venous return after regular dressing changes. Only two patients required a second operation, speciﬁcally skin grafting, to repair the secondary wound. The occurrence of localized skin petechiae and necrosis were caused by the destructive effects of ethanol on both the lesion and the return veins of the skin and subcutaneous tissue when destroying the deformity, which is a special phenomenon of sclerotherapy of IMVM. Among the two patients with venous embolism, one case was superﬁcial vein embolism, which showed local skin petechiae and necrosis and was cured after regular dressing changes, as shown in Figure 4. The other Frontiers in Surgery 05 frontiersin.org frontiersin.org Jin et al. 10.3389/fsurg.2023.1274313 FIGURE 2 Focal mass type IMVM in the right upper extremity. (A) The appearance before treatment. (B) The MRI before treatment. (C) The appearance after three times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgures were held by corresponding author. FIGURE 2 Focal mass type IMVM in the right upper extremity. (A) The appearance before treatment. (B) The MRI before treatment. (C) The appearance after three times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgures were held by corresponding author. case was a deep femoral vein embolism, characterized by swelling in the affected lower limb, the course of this condition lasted for more than 1 month, and the symptom gradually improved after anticoagulation treatment and wearing elastic stockings. Meanwhile, we observed that 39 patients had transient hematuria, all of which were hemoglobinuria. Some scholars believe that transient hematuria occurs after ethanol sclerotherapy due to the physiological reaction of the chemical nature of ethanol in the organism (9). frontiersin.org Frontiers in Surgery Discussion We have not observed severe hemolytic gross hematuria requiring intravenous application of binding bead protein. As far as coagulation is concerned, our previous studies have found elevated ﬁbrinogen degradation product and D–D in patients with ethanol sclerotherapy, and this elevation was often associated with a good therapeutic outcome; however, no serious disorders of coagulation were found (10). Among the late postoperative complications, a total of eight patients were observed, and ﬁve of them experienced muscle contracture, which was relieved in three patients after regular stretching of the contracted muscles and the corresponding rehabilitation treatment. However, two patients refused to receive further treatment, resulting in a deformity of the corresponding joint. Muscle contracture occurs due to muscle ﬁbrosis during ethanol sclerotherapy in IMVM, so patients should be treated at the early stage of contracture to prevent further ﬁbrosis and joint Frontiers in Surgery 06 frontiersin.org frontiersin.org Jin et al. 10.3389/fsurg.2023.1274313 FIGURE 3 Focal mass type IMVM in the left calf. (A) The appearance before treatment. (B) The MRI before treatment. The yellow ellipse indicated that the lesion was located in the gastrocnemius muscle. (C) The appearance after three times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgures were held by corresponding author. FIGURE 3 Focal mass type IMVM in the left calf. (A) The appearance before treatment. (B) The MRI before treatment. The yellow ellipse indicated that the lesion was located in the gastrocnemius muscle. (C) The appearance after three times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgures were held by corresponding author. FIGURE 3 Focal mass type IMVM in the left calf. (A) The appearance before treatment. (B) The MRI before treatment. The yellow ellipse indicated that the lesion was located in the gastrocnemius muscle. (C) The appearance after three times of treatment. (D) The MRI after treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgures were held by corresponding author. To minimize the incidence rate of complications caused by ethanol sclerotherapy, surgeons should be extremely careful during the operation. Prior to the procedure, fully understanding the anatomy of the puncture site is necessary to avoid unwanted damage to the arteries and important nerves. During the procedure, conﬁrming the puncture site could avoid leakage of sclerosant. Frontiers in Surgery frontiersin.org Discussion If the lesion is too small to conﬁrm the puncture in place, ﬂuoroscopy or ultrasound could assist. After the deformity. If the muscle contracture is severe and the conservative treatment fails, surgical intervention may be considered as a viable option, including excision of the diseased muscle or a Z-lengthening of the Achilles tendon, the Hoke technique, and the Taylor Spatial Frame external ﬁxation (11). Furthermore, three patients with peripheral nerve injury showed numbness in the innervated area. They were treated with neurotrophic therapy and completely recovered after 4–6 months. deformity. If the muscle contracture is severe and the conservative treatment fails, surgical intervention may be considered as a viable option, including excision of the diseased muscle or a Z-lengthening of the Achilles tendon, the Hoke technique, and the Taylor Spatial Frame external ﬁxation (11). Furthermore, three patients with peripheral nerve injury showed numbness in the innervated area. They were treated with neurotrophic therapy and completely recovered after 4–6 months. Frontiers in Surgery 07 frontiersin.org 10.3389/fsurg.2023.1274313 Jin et al. 10.3389/fsurg.2023.1274313 injection of anhydrous ethanol, it is necessary to perform ﬂuoroscopy or ultrasound procedures due to the highly destructive nature of anhydrous ethanol. Particularly for VMs in the facial or cervical part and deep tissue, operation under the premise of visualization has been found to improve the accuracy of sclerotherapy and perhaps reduce the occurrence of complications to a certain extent, which could improve the efﬁcacy of sclerotherapy. injection of anhydrous ethanol, it is necessary to perform ﬂuoroscopy or ultrasound procedures due to the highly destructive nature of anhydrous ethanol. Particularly for VMs in the facial or cervical part and deep tissue, operation under the premise of visualization has been found to improve the accuracy of sclerotherapy and perhaps reduce the occurrence of complications to a certain extent, which could improve the efﬁcacy of sclerotherapy. TABLE 3 Complications. n Percent (%) Intraoperative complications 51 43 Cough 47 40 Chess tightness 13 11 Allergic reaction 0 0 Early postoperative complications 58 49 Skin ulceration 15 13 Vein embolism 2 2 Hematuria 39 33 Infection 3 3 Heart failure 0 0 Coagulation disorder 0 0 Late postoperative complications 8 7 Muscle contracture 5 4 Joint deformity 2 2 Peripheral nerve injury 3 3 TABLE 3 Complications. Ethics statement The studies involving humans were approved by the Medical Ethics Committee of Jinling Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Written informed consent was obtained from the individual(s) for the publication of any potentially identiﬁable images or data included in this article. Acknowledgments The authors thank the participating patients and their families. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Funding Within safe doses, ethanol sclerotherapy effectively shrinks lesions, reduces pain, relieves motion limitation, and improves appearance, with minimal occurrence of severe complications. Overall, ethanol sclerotherapy for IMVM is safe and effective and could be the preferred treatment for IMVMs. However, it is crucial for surgeons to perform operations prudently and provide timely treatment for postoperative complications due to the potential occurrence of multiple short- or long-term complications. The authors declare ﬁnancial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Natural Science Foundation of China (No. 81272989, awarded to S-MY) and the “333 Project” of Jiangsu Province, China (No. BRA2020416, awarded to S-MY). 6. Bisdorff A, Mazighi M, Saint-Maurice JP, Chapot R, Lukaszewicz AC, Houdart E. Ethanol threshold doses for systemic complications during sclerotherapy of superﬁcial venous malformations: a retrospective study. Neuroradiology. (2011) 53(11):891–4. doi: 10.1007/s00234-010-0803-5 Author contributions (14, 15). In some huge and extensive inﬁltrative types of IMVMs, safe doses of sclerosants are often not enough for a good curative effect. Scholars tried to employ intravascular laser therapy for IMVM patients, in which the laser beams destroyed the lesion and the residual lesion was then treated with sclerotherapy. This kind of combined treatment also reduces the dose of sclerosants, thus decreasing the risks of complications or side effects. (14, 15). In some huge and extensive inﬁltrative types of IMVMs, safe doses of sclerosants are often not enough for a good curative effect. Scholars tried to employ intravascular laser therapy for IMVM patients, in which the laser beams destroyed the lesion and the residual lesion was then treated with sclerotherapy. This kind of combined treatment also reduces the dose of sclerosants, thus decreasing the risks of complications or side effects. C-JJ: Conceptualization, Investigation, and Writing – original draft. QW: Conceptualization, Data curation, and Writing – review and editing. MW: Formal analysis, Project administration, and Writing – review and editing. YC: Formal analysis, Project administration, and Writing – review and editing. S-MY: Conceptualization, Data curation, Methodology, Supervision, Writing – review and editing. 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. Discussion n Percent (%) Intraoperative complications 51 43 Cough 47 40 Chess tightness 13 11 Allergic reaction 0 0 Early postoperative complications 58 49 Skin ulceration 15 13 Vein embolism 2 2 Hematuria 39 33 Infection 3 3 Heart failure 0 0 Coagulation disorder 0 0 Late postoperative complications 8 7 Muscle contracture 5 4 Joint deformity 2 2 Peripheral nerve injury 3 3 To improve the effectiveness of ethanol sclerotherapy and reduce complications, researchers have developed ethanol gels. Because ethanol gel has a higher viscosity than liquid ethanol, it remains in the blood vessels for a longer period after injection into the vessels, resulting in a longer contact time with the lesion and a stronger effect on the local vessel wall. Thus, the ethanol dose and the occurrence of complications could be reduced, and the quality of life of the patients may be improved (12, 13). However, there are few clinical studies related to the use of ethanol gel, and additional research is required to conﬁrm its effectiveness for clinical application in treating IMVM. Furthermore, some scholars reported simultaneous application of physical and chemical treatment could effectively destroy the endothelium of malformed veins procedures, it is essential to closely monitor the vital signs of the patient and conduct regular follow-ups for early detection and early intervention of postoperative complications. Meanwhile, in order to avoid damaging normal veins and ensure the accurate FIGURE 4 Diffuse inﬁltrative type IMVM in the left foot. (A) The immediate postoperative appearance. The local skin showed extensively damaged. (B) The MRI before treatment, which indicated that the lesions were located in the plantaris muscle. (C) The appearance after 4 weeks of treatment. The local skin was intact with no scarring. (D) The MRI after three times of treatment. The yellow ellipse indicated the lesion. The copyrights of the ﬁgures were held by corresponding author. FIGURE 4 Diffuse inﬁltrative type IMVM in the left foot. (A) The immediate postoperative appearance. The local skin showed extensively damaged. (B) The MRI before treatment, which indicated that the lesions were located in the plantaris muscle. (C) The appearance after 4 weeks of treatment. The local skin was intact with no scarring. (D) The MRI after three times of treatment. The yellow ellipse indicated the lesion. 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https://openalex.org/W3165075628	https://headachemedicine.com.br/index.php/hm/article/download/362/760	es		As desventuras do paciente migranoso	Deleted Journal	2,013	cc-by	0
https://openalex.org/W3180368194	https://link.springer.com/content/pdf/10.1007/s40747-021-00445-3.pdf	Latin	null	Multi-objective particle swarm optimization with R2 indicator and adaptive method	Complex & intelligent systems	2,021	cc-by	9,649	Complex & Intelligent Systems (2021) 7:2697–2710 https://doi.org/10.1007/s40747-021-00445-3 Complex & Intelligent Systems (2021) 7:2697–2710 https://doi.org/10.1007/s40747-021-00445-3 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Multi-objective particle swarm optimization algorithms encounter significant challenges when tackling many-objective opti- mization problems. This is mainly because of the imbalance between convergence and diversity that occurs when increasing the selection pressure. In this paper, a novel adaptive MOPSO (ANMPSO) algorithm based on R2 contribution and adaptive method is developed to improve the performance of MOPSO. First, a new global best solutions selection mechanism with R2 contribution is introduced to select leaders with better diversity and convergence. Second, to obtain a uniform distribution of particles, an adaptive method is used to guide the flight of particles. Third, a re-initialization strategy is proposed to prevent particles from trapping into local optima. Empirical studies on a large number (64 in total) of problem instances have dem- onstrated that ANMPSO performs well in terms of inverted generational distance and hyper-volume metrics. Experimental studies on the practical application have also revealed that ANMPSO could effectively solve problems in the real world. Keywords Many-objective optimization problems · Multi-objective particle swarm optimization · R2 contribution · Adaptive strategy Multi‑objective particle swarm optimization with R2 indicator and adaptive method Qinghua Gu1,2 · Mengke Jiang1,2 · Song Jiang2,3 · Lu Chen1,2 Received: 6 November 2020 / Accepted: 10 June 2021 / Published online: 9 July 2021 © The Author(s) 2021 Introduction and car cab design with preference information [3]. Due to the conflicting characteristics of these objectives, it is difficult to find an optimal solution that fits all objectives. Therefore, it is obvious that the key to solve the MaOPs lies in finding a set of trade-off solutions [4, 5]. Many-objective optimization problems (MaOPs) are widely applied to many fields in the real world, such as water supply portfolio planning [1], crash safety design of vehicles [2], i gf To tackle MaOPs, many nature-inspired heuristic algo- rithms are proposed in recent years (NSGA-II [6], NSGA-III [7], MOPSO [8], and MOEA/D [9]). However, the effective- ness of these MOEAs deteriorate dramatically when dealing with MaOPs. Therefore, a number of redesigned MOEAs have sprung up, including RPD-NSGAII [10], NSGAII-SDR [11], RVEA [12], SRA [13], MOMBI-II [14] and IEA [15]. In addition to the MOEAs mentioned above, multi-objec- tive particle swarm optimization (MOPSO) attracts many people because of their highly competitive performance. Moreover, according to the results from [16], MOPSO is able to cover the full Pareto Front of the test functions and converge fast. In addition, the swarm intelligence algorithms like MOPSO are also useful in practical applications. For example, a hybrid discrete artificial bee colony algorithm is presented to solve a blocking lot-streaming flow shop (BLSFS) scheduling problem. This algorithm adopts three initialization strategies to improve the quality of the initial population [17]. An evolutionary multi-objective robust if To tackle MaOPs, many nature-inspired heuristic algo- rithms are proposed in recent years (NSGA-II [6], NSGA-III [7], MOPSO [8], and MOEA/D [9]). However, the effective- ness of these MOEAs deteriorate dramatically when dealing with MaOPs. Therefore, a number of redesigned MOEAs have sprung up, including RPD-NSGAII [10], NSGAII-SDR [11], RVEA [12], SRA [13], MOMBI-II [14] and IEA [15]. Mengke Jiang author contributed equally to this work. * Qinghua Gu qinghuagu@126.com Mengke Jiang mengkejiang2020@163.com Song Jiang jiangsong925@163.com Lu Chen LukeChen1993@outlook.com 1 School of Management, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China 2 Xi’an Key Laboratory for Intelligent Industrial Perception, Calculation and Decision, Xi’an University of Architecture and Technology, Xi’an 710055, China 3 School of Resources Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China Mengke Jiang author contributed equally to this work. Introduction * Qinghua Gu qinghuagu@126.com Mengke Jiang mengkejiang2020@163.com Song Jiang jiangsong925@163.com Lu Chen LukeChen1993@outlook.com 1 School of Management, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China 2 Xi’an Key Laboratory for Intelligent Industrial Perception, Calculation and Decision, Xi’an University of Architecture and Technology, Xi’an 710055, China 3 School of Resources Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China Mengke Jiang author contributed equally to this work. * Qinghua Gu qinghuagu@126.com Mengke Jiang mengkejiang2020@163.com Song Jiang jiangsong925@163.com Lu Chen LukeChen1993@outlook.com 1 School of Management, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China 2 Xi’an Key Laboratory for Intelligent Industrial Perception, Calculation and Decision, Xi’an University of Architecture and Technology, Xi’an 710055, China 3 School of Resources Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China Mengke Jiang author contributed equally to this work. In addition to the MOEAs mentioned above, multi-objec- tive particle swarm optimization (MOPSO) attracts many people because of their highly competitive performance. Moreover, according to the results from [16], MOPSO is able to cover the full Pareto Front of the test functions and converge fast. In addition, the swarm intelligence algorithms like MOPSO are also useful in practical applications. For example, a hybrid discrete artificial bee colony algorithm is presented to solve a blocking lot-streaming flow shop (BLSFS) scheduling problem. This algorithm adopts three initialization strategies to improve the quality of the initial population [17]. An evolutionary multi-objective robust 1 School of Management, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China 2 Xi’an Key Laboratory for Intelligent Industrial Perception, Calculation and Decision, Xi’an University of Architecture and Technology, Xi’an 710055, China 3 School of Resources Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, Shaanxi, China Vol.:(0123456789) 1 3 Complex & Intelligent Systems (2021) 7:2697–2710 2698 mainly use the Pareto-based strategy. However, the disad- vantage of these algorithms is that they may be unsuitable for tackling MaOPs. The main reason is that the gbest with poor performance impedes the convergence of the algorithm unavoidably. In light of this dilemma, the indicator-based strategy is suggested into MOPSOs, such as MOPSOhv [30], R2-MOPSO [31], R2SMMOPSO [32], IDMOPSO [33], and so on. The contributions of indicators are applied to select gbest in these algorithms. It is reported that desirable properties make the R2 indicator as a viable candidate to be incorporated into an indicator-based algorithm. Introduction There is a defect that some algorithms among them delete the external archive, which may lead to insufficient diversity. Meanwhile, the indicator of hyper-volume (HV) is likely to cause the increase of computational complexity. scheduling algorithm is proposed to tackle the new model of BLSFS [18]. The innovation of this algorithm is the sug- gestion of two novel crossover operations. The experimental studies have shown that the swarm intelligence algorithm is effective in solving practical problems. f However, the balance between convergence and diversity is still a particular issue to be addressed about MOPSOs. The convergence depends on the ability of particles to approach the true Pareto Front. Many algorithms increase selection pressure to improve convergence. Nevertheless, the diver- sity of solutions may decrease if the focus is only put on improving convergence. Thus, how to balance convergence and diversity for MOPSOs is a challenge. Many MOPSOs have been redesigned to deal with this problem. For exam- ple, a competitive mechanism is suggested in CMOPSO to improve diversity [19]. The domination-based strategy and decomposition-based strategy are combined in D2MOPSO, and it deletes the external archive [20]. A balanceable fit- ness estimation (BFE) method is proposed to improve both convergence and diversity in NMPSO, which updates the external archive by calculating the convergence distance and diversity distance [21]. The multi-group multi-objective col- laborative optimization framework proposed by Zhan et al. optimizes each objective function through sub-populations [22]. This algorithm sets up an archive maintenance mecha- nism based on Pareto to solve MOPs [22]. A “belief space” is developed to adjust the parameters of the velocity formula [23]. The random immigrants’ strategy is first introduced into MOPSOs to increase diversity and to prevent particles from falling into local optima [24]. These algorithms are proposed to balance convergence and diversity by improving diversity or the searching ability, which can increase the robustness of these algorithms. While, only increasing randomness cannot bring a qualitative leap to the overall performance of algo- rithms. Furthermore, it is difficult to ensure that convergence does not decrease while increasing diversity. Generally, these variant MOPSOs achieve promising results in solving problems with 2–3 objectives because of the outstanding heritages of PSO. There are a few approaches designed for MaOPs, these redesigned MOPSOs may suffer from effectiveness deterioration on the balance between convergence and diversity when solving MaOPs with complex Pareto Front. 1 3 1 3 (2) where i, j = 1, 2, …, m. In MaOP, if a solution is not domi- nated by any other solution, it can be referred to as a Pareto Optimal Solution. The corresponding objective vector set of the Pareto Set is called Pareto Front (PF). The balanceable fitness estimation (BFE) is proposed in [21]. This method is mainly used to define the fitness value of a particle by weights, which has a better performance when updating the external archive and selecting the global best solutions. Thus, this method is followed and combined with the R2 contribution in this paper. The main formula is as follows: Introduction The poor performance is mainly induced by the traditional gbest selection mechanism. Mean- while, a uniform distribution of solutions relies on the lead- ers and the searching ability of particles. Therefore, it is cru- cial to modify the flight of particles. Besides, most of them ignore the fact that the fast convergence rate may become a disadvantage of MOPSOs if it is not controlled effectively. f Motivated by these problems, this paper introduces the R2 contribution and an adaptive strategy into the novel multi- objective particle swarm (ANMPSO) to balance convergence and diversity. Three improvements are mainly proposed in this paper to respond to the current problems encountered by MOPSOs: 1. The global best selection solutions mechanism that uses the R2 contribution is introduced to select the gbest with better convergence and diversity. In fact, since the global best solution (gbest) and per- sonal best solution (pbest) guide the evolutionary search. The key of the balance between convergence and divergence lies in the performance of two leaders. According to this, some algorithms build a new mechanism of global best solutions selection. A new parallel coordinates system is proposed in [25]. This algorithm selects leaders by setting a leader group. A hybrid framework of the solution distri- bution entropy is adopted to select global best solutions in AMOPSO [26]. In addition, an adaptive strategy is used to update the flight parameters in this algorithm. The multi- competition leader selection is developed in a modified competitive mechanism multi-objective particle swarm [27]. The extreme solutions are proposed based on Pareto domi- nance in f-MOPSO/Div algorithm [28]. A framework of the grid-based ranking scheme is introduced to select leaders, which is designed for MaOPs [29]. These algorithms above 2. The adaptive strategy that uses the population spacing information to adjust parameters is proposed to improve the distribution of particles with appropriate diversity and convergence. 3. The updating of particle age is adopted to prevent parti- cles from trapping into local optima. The remaining parts of this paper are as follows. Some basic knowledge before this study is briefly described in the next section. The algorithm framework of ANMPSO and the details of each improvement are introduced in the third section. The experimental results compared with other algorithms are depicted in the fourth section. Finally, the conclusion and future works are described in the last section. Complex & Intelligent Systems (2021) 7:2697–2710 2699 Many‑objective problem (5) where W indicates a set of weight vectors. Each weight vec- tor w = (w1, w2,…, wM) ∈ W is placed in the M-dimensional objective space. 1 \|W\| denotes a probability distribution on W. For MaOPs, the weight vectors are randomly initialized in such a way that the sum of each weight vector is equal to one in the objective space. Without loss of generality, MaOP consists of more than three conflicting objectives [34]. A MaOP can be stated as (1) minimize F(퐱) = (f1(퐱), f2(퐱), ..., fm(퐱))T, subject to 퐱∈훀 (1) n the objective space. The contribution of a solution x ∈ A to the R2 indicator s defined as (6) CR2(퐱,퐀,W,z∗) = R2(퐀,W,z∗) −R2(퐀{퐱},W,z∗). where m is the number of objectives, x is the decision vector, and fi(x) is the ith objective function. The contribution of a solution x ∈ A to the R2 indicator is defined as If a decision vector y is dominated by another decision vector z, referred to as z dominates y or z ≺y , which can be expressed as (6) (2) ∀i∶fi(퐳) ≤fi(퐲) and ∃j∶fi(퐳) < fi(퐲), Basic knowledge (5) R2(퐀,W,z∗) = 1 \|W\| ∑ 휔∈W min x∈A { max i=1,2,...,M {wi(fi(x) −z∗ i )}} , Many‑objective particle swarm optimization (7) ﬁt(pi,P) = 훼× Cd(pi,P) + 훽× Cv(pi,P), (7) Many-objective particle swarm optimization (MOPSO) is pro- posed in [8]. In the evolutionary process, pbest represents the personal best solution, and gbest represents the global best solution. A gbest can be found by the whole particle swarm. In this paper, a new velocity updating equal is adopted [21]. In each iteration, the new velocity and the new position is updated by where Cd(pi,P) denotes the normalized diversity distances and Cv(pi,P) indicates the convergence distances of pi . α and β are two factors that mitigate the impact of diversity distances and convergence distances. Specific details can refer to [21]. (3) vi(t + 1) = wvi(t) + c1r1(xpbesti(t) −xi(t)) + c2r2(xgbesti −xi(t)) + c3r3(xgbesti −xpbesti), (4) xi(t + 1) = xi(t) + vi(t + 1), (3) vi(t + 1) = wvi(t) + c1r1(xpbesti(t) −xi(t)) + c2r2(xgbesti −xi(t)) + c3r3(xgbesti −xpbesti), (3) (4) The framework of ANMPSO is proposed in this section. Five parts are, respectively, described in the following sub- sections: select global best solutions, adaptive strategy, reinitialize, update external archive, and the evolutionary search on the external archive. Then, a complete algorithm of ANMPSO is proposed. ( ) (4) xi(t + 1) = xi(t) + vi(t + 1), where t represents the tth iteration in the evolutionary pro- cess; xpbesti and xgbesti are the positions of pbest and gbest; ω is the inertia weight; c1, c2, and c3 are learning factors; r1, r2, and r3 are the random values uniformly distributed in [0, 1]. 1 3 Balanceable fitness estimation (2) R2 indicator and R2 contribution The gbest selection mechanism is a critical step for the entire algorithm. The convergence, diversity, and effectiveness of the algorithm are affected by the selection of leaders. The BFE method assigns BFE value to each non-dominated solution in the external archive using the weight method. The gbest of this method is chosen equally from the top 10% of individuals in the external archive with better BFE values. To further improve diversity and convergence, R2 R2 indicator and R2 contribution are used to assess the rela- tive quality of two sets of individuals, which are proposed in [35, 36]. The Tchebycheff Scalarizing Function in MOEA/D is employed as the utility function in this paper [9].f Assuming that a standard weighted Tchebycheff function has a particular reference point z, the indicator can be used to assess the quality of a single individual set (A) against z: 1 3 1 3 2700 Complex & Intelligent Systems (2021) 7:2697–2710 in this paper to change the flying directions of particles. The dis- tance information between particles is used to build a non-linear function. The value of the distance between the adjacent particles and the ith particle at (t + 1)th iteration is defined as contribution is introduced in this paper. R2 contribution is to measure the relative quality of two approximations of the PF based on utility functions that map a vector ⃗y ∈ℜk to a scalar value u ∈ℜ. The utility function adopted by this paper is the Tcheby- cheff Function, which can be defined as (9) SP(t + 1) = √ √ √ √ 1 S −1 S ∑ i=1 (̄l(t + 1) −li(t + 1) )2, (9) (8) u(z) = u𝜔(⃗z) = − max i∈{1,2,...,M} 𝜔i\|\|z∗ i −zi\|\|, (8) where li (t + 1) is the minimum Manhattan distance between where li (t + 1) is the minimum Manhattan distance between the ith particle and the other particles, ̄l(t + 1) is the average minimum Manhattan distance of all particles. Here, a function is used to better describe the flight process of particles: where li (t + 1) is the minimum Manhattan distance between the ith particle and the other particles, ̄l(t + 1) is the average minimum Manhattan distance of all particles. The pseudo-code is set in Algorithm 1. The pseudo-code is set in Algorithm 1. (1 C1i(t + 1) = ⎧ ⎪ ⎨ ⎪⎩ C1i(t) SP(t + 1) = SP(t) C1i(t)𝜇(t + 1) SP(t + 1) > SP(t) C1i(t)(𝜇(t + 1) + 1) SP(t + 1) < SP(t) , (12) (12) (13) C2i(t + 1) = ⎧ ⎪ ⎨ ⎪⎩ C2i(t) SP(t + 1) = SP(t) C2i(t)𝜇(t + 1) SP(t + 1) > SP(t) C2i(t)(𝜇(t + 1) + 1) SP(t + 1) < SP(t) , (14) C3i(t + 1) = ⎧ ⎪ ⎨ ⎪⎩ C3i(t) SP(t + 1) = SP(t) C3i(t)𝜇(t + 1) SP(t + 1) > SP(t) C3i(t)(𝜇(t + 1) + 1) SP(t + 1) < SP(t) . (14) Algorithm Ⅱ Update parameters 1. ω--inertia weight, c1, c2, c3--learning factors; 2. for i=1 to N 3. calculate the distance between particles using (9); 4. use (9)-(13) to update ω, c1, c2, and c3. 5. end for Algorithm Ⅱ Update parameters Adaptive strategy Parameters are essential factors in the flying process of parti- cles. The parameters can directly affect the searching ability of particles by adjusting the flying speed and the directions of the particles. Better distribution is determined by the abilities of exploitation and exploration. Most existing works on MOPSOs show that the larger ω and c1, and the smaller c2 would lead to better global exploration. The opposite situation would promote the ability of local exploitation. To improve the distribution of particles, an adaptive method of adjusting parameters is proposed 1 3 R2 indicator and R2 contribution Here, a function is used to better describe the flight process of particles: where 휔= (휔1, ..., 휔k) ∈W is a weight vector, and z* is an ideal point (an objective vector that is not dominated by any point). R2 contribution can be calculated using the utility func- tion and the equal (6). According to the BFE value and R2 contribution to select leaders in the external archive. The detailed steps of the algorithm are described as follows: (10) 휇(t + 1) = e(SP(t+1)+0.8)−1−1, (10) where µ (t + 1) is the non-linear adaptive function of the flight process. Then, the proposed adaptive strategy for flight parameters ω, c1, c2, and c3 can be expressed by where µ (t + 1) is the non-linear adaptive function of the flight process. Then, the proposed adaptive strategy for flight parameters ω, c1, c2, and c3 can be expressed by 1. Calculate the BFE values of all the individuals in the external archive, and mark the top 10% of individuals with better BFE values. (11 𝜔i(t + 1) = ⎧ ⎪ ⎨ ⎪⎩ 𝜔i(t) SP(t + 1) = SP(t) 𝜔i (t)(𝜇(t + 1) + 1) SP(t + 1) > SP(t) 𝜔i(t)𝜇(t + 1) SP(t + 1) < SP(t) , 2. Calculate the R2 contributions of marked individuals. 3. Select the individuals which have the maximum contri- bution value of R2 and set as gbest. (11) Reinitialize In this paper, a regular dominance strategy is used to update pbest. pbest could be updated if the current position of parti- cle has dominated the pbest. Otherwise, the pbest keep con- stantly. Considering that the position of a particle remains 1 3 Complex & Intelligent Systems (2021) 7:2697–2710 2701 update the pbest based on Pareto. Line 20 is the process of updating the external archive using the BFE method. The evolutionary search strategy is applied on A to get a new swarm S, which provided another searching mode. At last the final solutions in A are reported as the Pareto Front. unchanged, it may fall into local optima. Thus, a re-initiali- zation strategy is proposed in this paper. The detailed steps are as follows: 1. The definition of a particle’s age is quoted and the initial value is set to 0. 2. The maximum value Ta is set to 2. 2. The maximum value Ta is set to 2. 3. If the pbest is not updated, the age of a particle would increase by one year. Once the age of a particle is over the value of Ta, the particle may trap into local optima. Equal (15) is used to do re-initialization in this situation: 3. If the pbest is not updated, the age of a particle would increase by one year. Once the age of a particle is over the value of Ta, the particle may trap into local optima. Equal (15) is used to do re-initialization in this situation: (15) xt+1 i ∼N (xgbesti−xpbesti 2 , \|xgbesti−xpbesti\| ) . (15) Update external archive The external archive of MOPSO has a threshold value. When the number of non-dominated solutions reaches this threshold value, the external archive must be updated. The BFE method is used in this paper to evaluate the qualities of the solutions by estimating the fitness value of the particles. When the archive needs to be updated, the solution with the smallest BFE value must be deleted. The external archive of MOPSO has a threshold value. When the number of non-dominated solutions reaches this threshold value, the external archive must be updated. The BFE method is used in this paper to evaluate the qualities of the solutions by estimating the fitness value of the particles. i When the archive needs to be updated, the solution with the smallest BFE value must be deleted. Evolutionary search on the external archive After the first few steps, some non-dominated solutions are remained in the external archive as leaders to guide the flight of other particles. To maximize the qualities of the solutions, the simulated binary crossover (SBX) and polynomial-based muta- tion (PM) are used in ANMPSO to perform an evolutionary search in the external archive. They are widely used in MOEAs to solve MOPs [22] and are expanded for MaOPs in this paper. Two searching modes are conducive to finding better solutions. Test functions In this section, a specific experimental process is discussed. DTLZ1–DTLZ7 [37] and WFG1–WFG9 [38] test functions with 4, 6, 8, and 10 objectives are used to evaluate the comprehension performance of ANMPSO. For DTLZ1–DTLZ7, the number of decision variables is set as D = m + k−1, where m represents the objective number. As suggested in [37], k = 5 for DTLZ1 and k = 10 for DTLZ2–DTLZ7. For WFG1–WFG9, the number of decision variables D is set as 24. Where the position-related parameters K = 2 × (m − 1), and distance-related parameters L = 20 [13, 21]. The complete algorithm of ANMPSO The previous five parts contain all the steps of ANMPSO, select global best solutions, adaptive strategy, reinitialize, update external archive, and evolutionary search on the external archive. The detailed pseudo-code of the algo- rithm is described in Algorithm 3. An initialization process is executed from line 1 to line 6. At the start of the opti- mization cycle, the velocities of particles are set to 0, and the positions are initialized randomly. Line 8 executes the selection of gbest, with details shown in Algorithm 1. Then, the algorithm steps into the main loop. Lines 12–13 are the processes of updating the velocities and the positions of par- ticles using an adaptive strategy. Lines 14–19 represent the re-initialization process. The main loop includes lines 9–16. After that, the dominance relationship will be checked to 1 3 Performance metrics The goal of the MaOPs is to minimize the distance between a set of solutions and true PF. To evaluate the performance of the algorithms under consideration, the comprehensive indicators of inverted generation distance (IGD) [39] and HV [40] are used in this paper. They are widely adopted in evaluating the performance of MOEAs [7, 41]. 1 3 1 3 2702 Complex & Intelligent Systems (2021) 7:2697–2710 A smaller IGD value means that the solutions are closer to the PF. The definition of IGD is into two archives: convergence-related and diversity-related namely. This algorithm could resolve MaOPs (up to 20 objectives) with satisfactory performance. (16) IGD = ∑̄N i=1 ̄di ̄N , IGD = ∑̄N i=1 ̄di ̄N , SRA [13]: SRA adopts the stochastic ranking technique to balance the searching biases of different indicators. It is a typical indicator-based algorithm. (16) where the ̄di indicates the Euclidean distance between the solution i in Pareto-optimal set to the nearest solution in P. The ̄N is the size of the Pareto-optimal set. Similar to [21], for calculating IGD, 200,000 reference points on the Pareto Front of each test instance are sampled by a system- atic approach [39, 42]. RPD-NSGAII [10]: RPD-NSGAII proposes a novel decomposition-based dominance relation to solve MaOPs, which could cope with MaOPs with up to 20 objectives. RPD-NSGAII [10]: RPD-NSGAII proposes a novel decomposition-based dominance relation to solve MaOPs, which could cope with MaOPs with up to 20 objectives. The individual parameter settings for each algorithm in this paper are set according to their respective papers. How- ever, due to the weight vector, the objective numbers are set the same as ANMPSO. As proposed in [40], the number of objectives is 165, 252, 330, and 275. The maximum number of evaluations in this paper is set to 105. These algorithms run 30 times to get the average values of IGD and HV. The HV indicator measures the volume solutions that are dominated by the approximate set. A larger HV value means a better performance of algorithm. The definition of HV is (17) HV(z∗,A) = L ({ ⋃ a∈A {b ∈Λ\|a ≺b ≺z∗} }) , Moreover, Wilcoxon’s sum test is implemented on the PlatEMO [46]. The results with a significance level of 0.05 obtained on the PlatEMO are used to analyze results. Performance metrics Where “+”, “-”, and “=” represent that the results obtained by other algorithms, respectively, mean “significantly better than”, “significantly worse than”, and “practically similar to” the one obtained by ANMPSO using this statistical test (Table 1). (17) where L is the Lebesgue measure, z* is the reference point in the objective space. According to [21], the z* is set as (1.0, 1.0, …, 1.0) for calculating HV. As recommended in [13], all the objectives are normalized using the vector 1.1 × (f1 max, f2 max, …, fm max), where fk max (k = 1, 2, …, m) is the maximum value of the kth objective in the true PF. Comparisons results on DTLZ1–DTLZ7 In this paper, seven state-of-the-art MOEAs (GrEA [43], RVEA [12], NMPSO [21], SPEA2-SDE [44], Two-Arch2 [45], SRA [13], and RPD-NSGAII [10]) are selected for a comparative experimental study. Each one is detailed as follows: Table 2 shows the IGD comparison results of ANMPSO for seven current MOEAs on DTLZ1–DTLZ7 with 4–10 objec- tives. As observed from Table 2, the proportion of the pro- posed ANMPSO algorithm in the test instances outperforms GrEA, RVEA, NMPSO, SPEA2-SDE, Two-Arch2, SRA, and RPD-NSGAII with a respective statistical significance of 12/28, 25/28, 24/28, 14/28, 10/28, 21/28, and 11/28. These results are summarized in the last row of Table 2. GrEA [43]: In GrEA, grid dominance that determines the mutual relationship of individuals is proposed to bal- ance convergence and diversity. Numerical studies show the typicality of this algorithm. RVEA [12]: RVEA uses reference vectors to decompose a multi-objective problem into several single-objective sub- problems. The term angle penalized distance is suggested to balance convergence and diversity. Experimental studies show that RVEA is highly competitive. The performances of algorithms vary not only with per- formance metrics but also with problem categories. For Table 1 parameters settings of all the algorithms compared Algorithms Parameters GrEA pc = 1, pm = 1/D, ηc = 20, ηm = 20 RVEA pc = 1, pm = 1/D, ηc = 30, ηm = 20, α = 2, fr = 0.1 NMPSO ω ∈ [0.1, 0.5], c1, c2, c3 ∈ [1.5, 2.5], pm = 1/D, ηm = 20 SPEA2-SDE pc = 0.9, pm = 1/D, ηc = 20, ηm = 20 Two-Arch2 p = 1/m, ηc = 15, ηm = 15 SRA pc = 1.0, pm = 0.1, ηc = 15, ηm = 15, pc ′ = 0.4 RPD-NSGAII pc = 1, pm = 1/D, ηc = 20, ηm = 20 ANMPSO pc = 1, pm = 1/D, ηc = 20, ηm = 20 Table 1 parameters settings of all the algorithms compared Table 1 parameters settings of all the algorithms compared Algorithms Parameters NMPSO [21]: NMPSO is a novel approach, which pro- poses the concept of balanced fitness value to increase the selection pressure and introduces the weighting method. NMPSO has a better performance in solving MaOPs com- pared with other MOPSOs. SPEA2-SDE [44]: To improve the convergence, SPEA2- SDE proposes the shifted distance estimation approach. Because of the particularity of this method, SPEA2-SDE is comparable. 1 3 1 3 Comparisons results on WFG1–WFG9 Tables 4 and 5 show the results of comparisons between ANMPSO and seven typical MOEAs on WFG1–WFG9 benchmark functions. From the results of Table 4, ANMPSO won 22 times out of 36 comparisons and won 21 times out of 36 comparisons on Table 5. According to the results in Table 4, regarding the WFG1 with a mixed front, ANMPSO is better than GrEA and SPEA2-SDE. Both RVEA and SRA have worse results. The WFG2 has a disconnect and convex front, ANMPSO out- performs other algorithms. The results of ANMPSO show a strong competitiveness for the WFG3 with a linear and degenerate front. The reason why ANMPSO performs well is that the adaptive strategy maintains the diversity in a high- dimensional space. The WFG4–9 problems have concave fronts. Among these comparative results, ANMPSO obtains the second-best results. In Table 3, the comparative results of ANMPSO and seven state-of-the-art algorithms are listed for DTLZ1–DTLZ7 with 4, 6, 8, and 10 objectives using HV. Similar to the conclusions observed from the IGD compari- son results, ANMPSO is the best-performing algorithm. Many compared algorithms cannot obtain the HV values when tackling DTLZ1 and DTLZ3 because they are multi- modal problems. According to HV metric values, it can be observed that ANMPSO performs better than other algo- rithms on DTLZ2 and DTLZ5 problems. From Table 5, the comparison results of ANMPSO and seven state-of-the-art algorithms are listed for WFG1–WFG9 using HV. Observed from the HV comparison of results, the advantages of ANMPSO are evident. ANMPSO obtains the best results on 21 out of 36 comparisons. While GrEA, NMPSO, SPEA2-SDE, Two-Arch2, and RPD-NSGAII, respectively, perform the best on 1, 1, 2, 3, and 8 compari- sons, RVEA and SRA do not have the best results. Figure 1 shows the curves of IGD metric values on DTLZ4 and DTLZ7 with 10 objectives. As can be seen from Fig. 1a, b, even if the curve changes of ANMPSO, NMPSO, and SPEA2-SDE are similar, ANMPSO has an obvious advantage over other algorithms and it obtains the best IGD values. As can be seen from Fig. 1b, although the curve of RVEA shows a rapid downward trend in the early stage, it begins to rise slightly in the later stage. Comparisons results on DTLZ1–DTLZ7 1 Variation of median IGD metric values of all algorithms for DTLZ4 (a) and DTLZ7 (b) test suits using 10 objectives (a) DTLZ4 (b) DTLZ7 Fig. 1 Variation of median IGD metric values of all algorithms for DTLZ4 (a) and DTLZ7 (b) test suits using 10 objectives Fig. 1 Variation of median IGD metric values of all algorithms for DTLZ4 (a) and DTLZ7 (b) test suits using 10 objectives DTLZ1 and DTLZ3 problems, GrEA and RPD-NSGAII achieve the best IGD values, respectively. ANMPSO ranks 3rd, as it outperforms other algorithms. For the DTLZ2 problem, ANMPSO is superior than RVEA, NMPSO, SPEA2-SDE, and SRA, has achieved moderate performance of ANMPSO. The curves of SPEA2-SDE and NMPSO are barely fluctuated during the later iteration. This situation demonstrates that these algorithms probably fall into local optima. Generally, by comparing the results, ANMPSO has excellent performance compared these MOEAs. Fig. 1 Variation of median IGD metric values of all algorithms for DTLZ4 (a) and DTLZ7 (b) test suits using 10 objectives (a) DTLZ4 (b) DTLZ7 Fig. 1 Variation of median IGD metric values of all algorithms for DTLZ4 (a) and DTLZ7 (b) test suits using 10 objectives (a) DTLZ4 (b) DTLZ7 (a) DTLZ4 (b) DTLZ7 (b) DTLZ7 of ANMPSO. The curves of SPEA2-SDE and NMPSO are barely fluctuated during the later iteration. This situation demonstrates that these algorithms probably fall into local optima. Generally, by comparing the results, ANMPSO has excellent performance compared these MOEAs. DTLZ1 and DTLZ3 problems, GrEA and RPD-NSGAII achieve the best IGD values, respectively. ANMPSO ranks 3rd, as it outperforms other algorithms. For the DTLZ2 problem, ANMPSO is superior than RVEA, NMPSO, SPEA2-SDE, and SRA, has achieved moderate performance in terms of IGD. For the DTLZ4 problem with large “hill sizes”, the main challenge of maintaining diversity of the population is caused by a non-uniform density of solutions. Many algorithms cannot jump out of the local optima for DTLZ4. ANMPSO performs best for IGD in this problem because that the re-initialization strategy can prevent par- ticles from falling local optima. For the DTLZ5 problem, Two-Arch2 ranks 1st, and SRA is second only to Two- Arch2. Although this problem is degenerate, ANMPSO has the best performance, indicating that ANMPSO can solve problems with complex true PF. For the DTLZ6 prob- lem, ANMPSO beats GrEA, RVEA, and SRA. Its perfor- mance is similar to SPEA2-SDEs. Comparisons results on DTLZ1–DTLZ7 For the DTLZ7 problem, ANMPSO also shows a superior performance than other compared algorithms. 1 3 Comparisons results on DTLZ1–DTLZ7 Two-Arch2 [45]: To balance the convergence, diversity, and complexity in solving MaOPs, particles are divided 2703 Complex & Intelligent Systems (2021) 7:2697–2710 Table 2 Comparison results of ANMPSO and seven MOEAs on DTLZ1–7 using IGD Problem M GrEA RVEA NMPSO SPEA2-SDE Two-Arch2 SRA RPD-NSGAII ANMPSO DTLZ1 4 9.8494e−2(5.82e−2)+ 4.2733e−1(2.79e−1)− 2.8651e+0(2.13e+0)− 2.6561e−1(2.09e−1)+ 3.7521e−2(6.04e−4)+ 6.8833e−1(3.21e−1) - 4.4442e−2(1.08e−3)+ 3.3441e−1(1.49e−1) 6 2.0326e−1(7.27e−2)+ 3.9546e−1(2.58e−1)− 2.3276e+0(2.31e+0)− 5.8283e−1(2.65e−1)+ 6.5804e−2(9.18e−4)+ 1.5773e+0(7.37e−1)− 7.4408e−2(1.47e−3)+ 2.4179e−1(9.14e−2) 8 2.6257e−1(6.24e−2)+ 3.5698e−1(2.04e−1)+ 7.7916e+0(9.51e+0)− 8.5971e−1(2.58e−1)− 8.9460e−2(1.21e−3)+ 2.3688e+0(8.58e−1)− 1.1586e−1(1.48e−2)+ 4.8711e−1(7.58e−1) 10 3.6822e−1(3.96e−2)+ 3.3964e−1(2.04e−1)+ 1.3653e+1(1.40e+1)− 6.0556e−1(2.37e−1)− 1.0937e−1(2.24e−3)+ 1.5647e+0(5.74e−1)− 1.6935e−1(1.77e−2)+ 5.8575e−1(7.43e−1) DTLZ2 4 1.1865e−1(1.54e−3)= 1.4101e−1(3.12e−4)− 1.5675e−1(3.29e−3)− 1.5822e−1(2.67e−3)− 1.1280e−1(1.40e−3)− 1.5616e−1(3.69e−3)− 1.6057e−1(5.64e−3)− 1.0830e−1(9.75e−4) 6 2.2021e−1(9.48e−4)− 2.8168e−1(1.15e−3)− 2.8573e−1(1.99e−2)− 2.9467e−1(2.02e−3)− 2.3022e−1(1.83e−3)− 2.8885e−1(3.79e−3)− 3.2207e−1(2.13e−2)− 2.1495e−1(8.69e−4) 8 3.1434e−1(3.35e−3)+ 3.8850e−1(2.41e−3)− 3.9149e−1(3.84e−2)− 3.9538e−1(3.60e−3)− 3.3431e−1(2.79e−3)+ 3.9263e−1(4.89e−3)− 3.1344e−1(6.86e−4)+ 3.4811e−1(5.54e−2) 10 4.0660e−1(9.18e−4)+ 5.2150e−1(1.55e−2)− 5.3852e−1(8.18e−2)− 5.0125e−1(4.31e−3)− 4.1671e−1(3.49e−3)+ 4.7953e−1(7.18e−3)− 4.3599e−1(3.53e−3)+ 4.5765e−1(9.73e−2) DTLZ3 4 6.6510e−1(2.71e−1)+ 1.5188e+1(6.21e+0)− 9.7355e+1(1.82e+1)− 1.0359e+1(4.48e+0)+ 1.1844e−1(8.62e−3)+ 2.2620e+1(1.32e+1)− 1.3773e−1(3.56e−3)+ 1.1225e+1(1.68e+1) 6 7.4738e−1(2.05e−1)+ 1.2549e+1(5.25e+0)− 1.0263e+2(2.74e+1)− 1.9657e+1(1.17e+1)+ 3.0369e−1(2.19e−2)+ 3.9338e+1(2.20e+1)− 2.5829e−1(9.08e−3)+ 9.2709e−1(2.57e+1) 8 3.7797e+0(1.54e+0)+ 1.2573e+1(5.92e+0)− 1.2518e+2(4.33e+1)− 2.5415e+1(1.85e+1)− 6.8307e−1(3.29e−1)+ 6.0265e+1(3.90e+1)− 3.7507e−1(1.11e−1)+ 1.0070e+1(2.93e+1) 10 6.0561e+0(3.03e+0)+ 1.7844e+1(1.07e+1)− 1.4088e+2(5.46e+1)− 2.1286e+1(1.39e+1)− 1.1568e+1(6.45e−1)+ 5.9504e+1(3.28e+1)− 4.8433e−1(2.16e−2)+ 1.3847e+1(3.59e+1) DTLZ4 4 1.5543e−1(1.02e−1)− 1.5183e−1(5.77e−2)− 1.7087e−1(7.80e−2)− 1.8839e−1(1.43e−1)− 1.1255e−1(1.35e−3)− 1.3546e−1(5.46e−3)− 1.6853e−1(3.73e−3)− 1.0876e−1(1.15e−3) 6 2.2109e−1(1.09e−3)+ 3.5475e−1(9.62e−2)− 3.6319e−1(1.12e−1)− 2.4304e−1(9.29e−3)+ 2.3393e−1(2.72e−3)+ 2.4600e−1(1.02e−2)+ 2.1840e−1(2.19e−3)+ 2.5783e−1(1.31e−1) 8 3.1885e−1(5.47e−3)= 4.5452e−1(6.21e−2)− 4.9625e−1(8.19e−2)− 3.1642e−1(2.71e−2)= 3.4125e−1(3.51e−3)− 3.3656e−1(2.58e−2)− 5.4499e−1(6.80e−2)− 3.1433e−1(1.37e−3) 10 4.0983e−1(7.47e−4)= 5.9589e−1(5.69e−2)− 5.3639e−1(7.24e−2)− 4.2923e−1(1.58e−2)− 4.1445e−1(3.12e−3)− 4.3739e−1(1.25e−2)− 4.4331e−1(1.52e−3)− 4.0741e−1(9.16e−2) DTLZ5 4 7.7406e−2(1.60e−2)− 1.8375e−2(3.16e−2)− 3.0404e−2(4.03e−3)+ 3.2914e−2(4.47e−3)+ 3.5852e−2(4.25e−3)+ 2.7019e−2(5.75e−3)+ 8.4522e−2(1.58e−2)− 4.0491e−2(5.55e−3) 6 2.1992e−1(4.79e−2)− 3.2540e−1(9.25e−2)− 2.2158e−1(2.10e−1)= 8.3483e−2(1.16e−2)+ 7.3858e−2(1.00e−2)+ 7.8672e−2(1.26e−2)+ 9.1905e−2(1.11e−2)+ 1.8323e−1(1.37e−1) 8 2.3803e−1(6.56e−2)+ 3.6554e−1(1.81e−1)= 4.6336e−1(2.02e−1)− 1.1720e−1(1.90e−2)+ 1.1241e−1(1.83e−2)+ 1.0577e−1(1.58e−2)+ 1.1325e−1(1.28e−2)+ 3.6552e−1(2.39e−1) 10 2.6610e−1(5.71e−2)− 3.5064e−1(2.00e−1)− 2.3652e−1(2.11e−1)− 1.3570e−1(2.46e−2)+ 1.5615e−1(2.91e−2)+ 1.1574e−1(1.69e−2)+ 1.5519e−1(2.09e−2)+ 2.0278e−1(1.71e−1) DTLZ6 4 1.8865e−1(5.67e−2)+ 2.7861e−1(2.89e−1)− 2.4133e−2(2.95e−3)+ 6.0509e−2(1.77e−2)+ 4.5803e−2(7.54e−3)+ 7.3707e−2(6.17e−2)+ 4.0660e−1(2.53e−1)− 2.0991e−1(6.32e−1) 6 3.4076e−1(4.83e−2)− 4.3410e−1(3.68e−1)− 7.5282e−2(1.86e−2)+ 1.5518e−1(5.38e−2)+ 1.2187e−1(2.25e−2)+ 4.3522e−1(4.99e−1)− 2.3152e−1(2.09e−1)+ 2.4026e−1(8.71e−1) 8 3.8700e−1(8.06e−2)− 6.6205e−1(3.75e−1)− 6.5160e−1(2.02e−1)− 2.0620e−1(1.36e−1)+ 1.9580e−1(6.61e−2)+ 8.7670e−1(6.80e−1)− 1.7936e−1(1.13e−1)+ 2.3317e−1(8.90e−1) 10 9.5077e−1(3.17e−1)− 4.5961e−1(3.18e−1)− 7.3710e−1(2.74e−2)− 2.4974e−1(1.14e−1)− 3.9296e−1(1.03e−1)− 1.4751e+0(1.04e+0)+ 1.5873e−1(4.61e−2)+ 2.4564e−1(7.94e−1) DTLZ7 4 1.5639e−1(6.06e−3)− 2.4670e−1(9.15e−3)− 1.2803e−1(8.46e−3)− 1.1844e−1(2.95e−3)+ 1.4952e−1(6.31e−2)− 1.4552e−1(6.51e−3)− 2.0643e−1(7.68e−2)− 1.3103e−1(8.45e−1) 6 2.8166e−1(5.57e−3)− 7.0226e−1(2.70e−2)− 3.0078e−1(3.10e−2)− 2.8646e−1(2.33e−2)− 3.3799e−1(3.03e−2)− 3.1008e−1(5.99e−3)− 4.7331e−1(1.74e−2)− 2.4979e−1(2.06e−1) 8 5.7852e−1(2.31e−2)− 1.8079e+0(2.03e−1)− 5.7413e−1(1.34e−1)− 5.9704e−1(3.50e−3)− 5.1254e−1(3.86e−3)− 5.7236e−1(4.88e−3)− 1.0425e+0(4.44e−2)− 5.0547e−1(7.53e−1) 10 2.5665e+0(9.97e−2)− 1.9436e+0(4.96e−1)− 9.3411e−1(3.64e−1)− 8.5332e−1(4.30e−2)− 7.8876e−1(1.50e−2)− 8.4070e−1(5.07e−3)− 1.5203e+0(1.85e−1)− 6.5139e−1(1.07e−1) ±/= 14/12/2 2/25/1 3/24/1 13/14/1 18/10/0 7/21/0 17/11/0 2704 Complex & Intelligent Systems (2021) 7:2697–2710 Table 3 Comparison results of ANMPSO and seven MOEAs on DTLZ1–7 using HV Problem M GrEA RVEA NMPSO SPEA2-SDE Two-Arch2 SRA RPD-NSGAII ANMPSO DTLZ1 4 7.1692e−1(1.07e−1)+ 3.8262e−1(3.89e−1)+ 0.0000e+0(0.00e+0)= 4.7835e−1(3.65e−1)+ 9.4192e−1(6.40e−4)+ 0.0000e+0(0.00e+0)= 9.3620e−1(1.18e−3)+ 0.0000e+0(0.00e+0) 6 7.8835e−1(1.70e−1)+ 2.8764e−1(3.92e−1)+ 0.0000e+0(0.00e+0)= 2.5675e−1(3.17e−1)+ 9.8873e−1(4.77e−4)+ 0.0000e+0(0.00e+0)= 9.8935e−1(4.47e−4)+ 0.0000e+0(0.00e+0) 8 7.3687e−1(2.52e−1)+ 2.7478e−1(2.13e−1)+ 0.0000e+0(0.00e+0)= 6.7907e−3(1.69e−2)= 9.9540e−1(3.66e−4)+ 0.0000e+0(0.00e+0)= 9.8909e−1(1.10e−2)+ 0.0000e+0(0.00e+0) 10 5.3943e−1(1.53e−1)+ 3.6681e−1(4.43e−1)+ 0.0000e+0(0.00e+0)= 7.6304e−2(1.10e−1)= 9.9591e−1(5.02e−4)+ 0.0000e+0(0.00e+0)= 9.6432e−1(1.64e−2)+ 6.0616e−2(1.71e−1) DTLZ2 4 7.1183e−1(4.70e−4)− 6.8498e−1(9.23e−4)− 6.9368e−1(3.65e−3)− 6.9728e−1(1.47e−3)− 6.9372e−1(4.03e−3)− 6.7206e−1(2.90e−3)− 7.1157e−1(1.68e−3)− 7.9244e−1(1.58e−3) 6 8.6988e−1(7.70e−4)− 8.1220e−1(1.52e−3)− 8.4722e−1(1.09e−2)− 8.4396e−1(2.04e−3)− 8.0839e−1(4.78e−3)− 7.8965e−1(3.73e−3)− 8.7327e−1(8.35e−4)− 8.9755e−1(1.83e−1) 8 9.0906e−1(1.81e−3)− 8.5446e−1(1.29e−2)− 9.0951e−1(5.23e−3)− 9.1220e−1(2.12e−3)− 8.2792e−1(7.99e−3)− 8.0205e−1(2.64e−2)= 9.4233e−1(7.71e−4)− 9.8901e−1(3.03e−1) 10 9.7061e−1(3.69e−4)+ 9.3127e−1(3.11e−3)+ 9.2908e−1(1.72e−2)= 9.4365e−1(4.25e−3)+ 7.8436e−1(1.05e−2)− 8.2686e−1(1.72e−2)− 9.6889e−1(6.52e−4)+ 9.3020e−1(4.07e−2) DTLZ3 4 2.6045e−1(1.64e−1)+ 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 6.9178e−1(1.00e−2)+ 0.0000e+0(0.00e+0)= 6.8731e−1(3.01e−3)+ 0.0000e+0(0.00e+0) 6 3.2980e−1(1.42e−1)+ 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 7.6346e−1(2.65e−2)+ 0.0000e+0(0.00e+0)= 8.4666e−1(9.24e−3)+ 0.0000e+0(0.00e+0) 8 6.6870e−3(1.77e−2)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 4.7434e−1(2.38e−1)+ 0.0000e+0(0.00e+0)= 9.0613e−1(1.16e−2)+ 0.0000e+0(0.00e+0) 10 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 1.4121e−1(1.72e−1)= 0.0000e+0(0.00e+0)= 9.3255e−1(2.04e−2)+ 0.0000e+0(0.00e+0) DTLZ4 4 6.8679e−1(6.28e−2)− 6.8510e−1(8.06e−4)− 6.8766e−1(3.93e−3)− 6.4062e−1(1.40e−1)− 6.9502e−1(1.04e−3)− 6.9578e−1(4.00e−3)− 7.1249e−1(9.56e−4)− 7.8446e−1(4.49e−3) 6 8.7020e−1(5.63e−4)= 8.0626e−1(2.29e−2)− 8.1004e−1(3.00e−2)− 8.7155e−1(8.76e−4)− 7.7889e−1(4.49e−3)− 8.4776e−1(4.71e−3)− 8.7189e−1(8.82e−4)− 8.7201e−1(3.25e−2) 8 9.1428e−1(4.09e−3)− 8.4414e−1(3.93e−2)− 8.5352e−1(2.10e−2)− 9.4050e−1(1.81e−3)+ 8.0667e−1(6.46e−3)− 9.1705e−1(4.40e−3)− 9.4451e−1(1.29e−3)+ 9.2450e−1(2.72e−2) 10 9.7123e−1(2.22e−4)+ 9.1164e−1(3.91e−2)− 9.4838e−1(7.41e−3)− 9.6674e−1(1.49e−3)+ 7.9282e−1(9.89e−3)− 9.4467e−1(4.38e−3)− 9.5086e−1(8.70e−4)− 9.5753e−1(1.77e−1) DTLZ5 4 1.1835e−1(6.50e−3)− 1.0626e−1(8.40e−3)− 1.3590e−1(2.41e−3)− 1.4032e−1(1.47e−3)= 1.4003e−1(1.82e−3)= 1.3628e−1(1.92e−3)− 1.2937e−1(6.89e−3)− 1.4466e−1(4.70e−3) 6 8.7300e−2(2.68e−3)− 9.1418e−2(2.10e−3)− 4.2179e−2(3.52e−2)− 9.6114e−2(2.72e−3)− 1.0581e−1(1.99e−3)= 5.4155e−2(1.42e−2)− 1.0564e−1(1.46e−3)− 1.0540e−1(2.24e−2) 8 7.9421e−2(7.65e−3)− 9.1995e−2(2.58e−3)− 2.3440e−2(2.63e−2)− 8.3318e−2(8.06e−3)− 9.4576e−2(1.56e−3)− 2.8480e−2(2.31e−2)− 9.4891e−2(1.50e−3)− 9.6800e−2(1.20e−2) 10 1.0651e−2(2.28e−2)+ 9.1364e−2(1.13e−3)+ 7.0622e−3(1.27e−2)= 8.2296e−2(5.76e−3)+ 9.1962e−2(7.45e−4)+ 2.3231e−2(1.54e−2)+ 9.2868e−2(4.18e−4)+ 0.0000e+0(0.00e+0) DTLZ6 4 1.0558e−1(7.10e−3)− 9.3154e−2(2.33e−2)− 1.4281e−1(1.48e−3)− 1.2844e−1(7.46e−3)− 1.3891e−1(1.61e−3)− 1.2372e−1(2.06e−2)− 5.9510e−2(5.55e−2)− 1.5378e−1(5.39e−2) 6 9.0903e−2(1.44e−4)+ 5.8773e−2(4.31e−2)− 1.0693e−1(3.65e−3)+ 8.1212e−2(3.56e−2)− 1.0116e−1(2.06e−3)+ 3.6472e−2(4.32e−2)− 8.8118e−2(3.90e−2)− 9.4571e−2(3.86e−2) 8 8.9748e−2(5.97e−3)+ 6.5548e−2(4.48e−2)− 9.1591e−2(1.80e−3)+ 7.7730e−2(3.03e−2)− 9.3755e−2(1.77e−3)+ 3.3903e−4(8.94e−4)− 9.6761e−2(1.22e−3)+ 8.2987e−2(3.44e−2) 10 0.0000e+0(0.00e+0)− 6.4002e−2(4.38e−2)− 9.0909e−2(0.00e+0)− 6.2409e−2(4.25e−2)− 9.1019e−2(2.42e−4)− 1.7624e−6(3.43e−6)− 9.4006e−2(3.50e−4)+ 9.2987e−2(3.44e−2) DTLZ7 4 2.7224e−1(2.13e−3)− 2.4836e−1(3.44e−3)− 2.8036e−1(3.79e−3)= 2.8485e−1(1.01e−3)= 2.7806e−1(1.86e−3)− 2.8236e−1(7.01e−4)− 2.6820e−1(2.50e−4)− 2.8521e−1(9.62e−2) 6 2.5535e−1(2.33e−3)= 1.9571e−1(8.36e−4)− 2.6004e−1(7.83e−3)− 2.3802e−1(5.15e−3)− 2.0599e−1(1.14e−2)− 2.4995e−1(1.37e−3)− 2.3001e−1(1.12e−3)− 2.6385e−1(7.51e−2) 8 2.1679e−1(2.59e−3)+ 1.6647e−1(5.63e−3)− 2.3441e−1(1.52e−2)+ 2.0684e−1(1.05e−2)= 1.8841e−1(1.62e−2)− 1.8257e−1(1.23e−2)− 1.8859e−1(3.16e−3)− 2.0838e−1(3.49e−2) 10 2.1810e−1(5.64e−3)+ 1.3787e−1(1.64e−2)+ 1.9765e−1(1.95e−2)+ 4.2055e−2(1.14e−2)− 9.3264e−2(3.72e−2)− 8.2260e−2(3.39e−2)− 1.7343e−1(3.86e−3)+ 1.2586e−1(1.52e−2) ±/= 13/11/4 6/18/4 5/13/10 6/16/6 10/15/3 1/18/9 14/14/0 Complex & Intelligent Systems (2021) 7:2697–2710 2705 Fig. Comparisons results on WFG1–WFG9 The curve’s trend of Two-Arch2 shows that the convergence speed of this algorithm is slow, and the IGD value is worse than that Figures 2 and 3 show parallel coordinates of the solu- tion sets on the WFG3 and WFG5 for 10 objectives with the best IGD values among all the 30 runs and 105 evalua- tions. In two figures, each polyline in the graph represents a solution, and each vertex on the polyline represents an objective value. The WFG3 has a linear and convex front to detect whether MOEAs have better performances. It can be observed from Fig. Comparisons results on WFG1–WFG9 2 that ANMPSO can completely 1 3 1 3 2706 Complex & Intelligent Systems (2021) 7:2697–2710 Table 4 Comparison results of ANMPSO and seven MOEAs on WFG1–9 using IGD roblem M GrEA RVEA NMPSO SPEA2-SDE Two-Arch2 SRA RPD-NSGAII ANMPSO WFG1 4 3.7976e−1(5.68e−3)− 1.4097e+0(2.02e−1)− 1.8300e+0(2.11e−1)− 2.9514e−1(8.97e−3)− 2.3899e−1(3.91e−3)− 1.5564e+0(1.69e−1)− 3.0047e−1(8.61e−3)− 2.0115e−1(3.03e−2) 6 6.7697e−1(3.82e−3)− 1.8481e+0(2.24e−1)− 2.3529e+0(1.86e−1)− 5.3603e−1(2.27e−2)− 5.0454e−1(2.13e−2)− 2.0761e+0(1.29e−1)− 4.9171e−1(6.45e−3)− 4.3238e−1(7.26e−3) 8 1.2175e+0(4.09e−2)− 2.2588e+0(1.37e−1)− 3.0264e+0(3.62e−1)− 8.6376e−1(2.82e−2)+ 1.0070e+0(3.27e−2)= 2.6352e+0(2.33e−2)− 8.5281e−1(3.59e−2)+ 1.0574e+0(5.17e−2) 10 1.0969e+0(1.73e−2)= 2.1988e+0(1.94e−1)− 2.7577e+0(2.50e−1)− 1.0818e+0(4.14e−2)− 1.1529e+0(8.46e−2)= 2.8977e+0(1.42e−1)− 1.0155e+0(2.38e−2)+ 1.1331e+0(5.33e−2) WFG2 4 3.8364e−1(1.42e−2)− 3.6769e−1(1.16e−2)− 5.3405e−1(4.92e−2)− 3.9184e−1(2.22e−2)− 2.5989e−1(3.89e−3)+ 5.3488e−1(2.51e−1)− 2.6542e−1(5.96e−3)= 2.6665e−1(2.96e−3) 6 5.9466e−1(2.65e−2)− 6.9172e−1(1.39e−2)− 1.1619e+0(1.88e−1)− 6.1340e−1(2.11e−2)− 4.8775e−1(3.69e−3)+ 7.0757e−1(1.74e−2)− 5.1860e−1(1.50e−2)= 5.1808e−1(5.07e−2) 8 1.0492e+0(4.25e−2)− 1.1855e+0(4.96e−2)− 1.5865e+0(1.33e−1)− 9.5664e−1(1.61e−2)− 7.7479e−1(5.93e−3)− 7.5329e−1(9.11e−2)= 9.2223e−1(1.77e−2)− 7.5412e−1(1.56e−2) 10 1.1904e+0(2.97e−2)− 1.1133e+0(5.94e−2)= 1.8447e+0(2.84e−1)− 1.1409e+0(3.60e−2)− 1.7398e+0(1.53e−2)− 1.2876e+0(7.20e−2)− 1.1052e+0(7.19e−3)− 1.0982e+0(9.71e−1) WFG3 4 2.0252e−1(1.86e−2)− 3.9568e−1(3.10e−2)− 2.0268e−1(1.83e−2)− 2.7647e−1(5.24e−2)− 1.8934e−1(1.07e−2)− 4.2579e−1(4.03e−2)− 2.8953e−1(2.72e−2)− 1.5189e−1(3.48e−2) 6 4.6065e−1(5.58e−2)= 1.3225e+0(5.38e−1)− 5.9150e−1(4.27e−2)− 6.3588e−1(1.63e−1)− 5.1158e−1(5.75e−2)− 1.2711e+0(5.93e−1)− 5.5714e−1(5.34e−2)− 3.0760e−1(9.34e−2) 8 6.7276e−1(1.00e−1)− 2.4413e+0(8.23e−1)− 1.0483e+0(2.08e−1)− 9.2118e−1(1.64e−1)− 7.9152e−1(2.25e−2)− 1.5851e+0(1.86e−1)− 9.5172e−1(6.67e−2)− 3.3578e−1(3.86e−2) 10 1.8355e+0(1.81e−1)− 4.2576e+0(1.72e+0)− 1.1036e+0(1.84e−1)− 1.5448e+0(3.14e−1)− 1.2993e+0(7.99e−2)− 1.8562e+0(2.49e−1)− 1.5569e+0(9.11e−3)− 5.1244e−1(4.97e−2) WFG4 4 5.6209e−1(3.01e−3)+ 6.9844e−1(9.75e−4)+ 7.5024e−1(1.63e−2)− 6.7594e−1(2.36e−2)= 5.3504e−1(4.11e−3)+ 7.6953e−1(6.70e−3)− 5.5246e−1(2.59e−3)+ 7.0070e−1(2.60e−2) 6 1.5234e+0(3.00e−3)− 1.9541e+0(1.03e−2)− 1.9724e+0(4.00e−2)− 1.6696e+0(1.52e−2)− 1.4963e+0(9.05e−3)= 1.9631e+0(3.85e−2)− 1.5122e+0(2.35e−3)− 1.4619e+0(7.12e−2) 8 2.6822e+0(1.56e−2)= 3.5546e+0(6.57e−2)− 3.3462e+0(6.35e−2)− 2.7460e+0(1.91e−2)− 2.7461e+0(3.06e−2)− 3.4659e+0(5.23e−2)− 2.8087e+0(1.28e−2)− 2.6705e+0(2.18e−1) 10 4.1156e+0(2.74e−2)+ 6.0213e+0(6.92e−2)− 5.0596e+0(3.40e−2)− 4.4589e+0(2.56e−2)= 4.2494e+0(3.29e−2)= 5.3071e+0(7.28e−2)− 4.6060e+0(9.60e−3)− 4.4038e+0(1.47e+0) WFG5 4 5.6999e−1(6.08e−3)= 6.9085e−1(6.48e−4)− 5.4170e−1(1.07e−2)= 6.9608e−1(5.10e−3)− 5.4569e−1(1.03e−2)− 7.7072e−1(2.68e−2)− 5.5065e−1(1.49e−2)− 5.4042e−1(1.69e−2) 6 1.4323e+0(7.67e−3)− 1.9581e+0(2.80e−2)− 1.8932e+0(2.86e−2)− 1.6824e+0(2.25e−2)− 1.4884e+0(1.52e−2)− 1.9500e+0(5.50e−2)− 1.4867e+0(4.09e−3)− 1.3160e+0(2.13e−2) 8 2.5622e+0(1.23e−2)= 3.7296e+0(5.62e−2)− 3.3004e+0(5.02e−2)− 2.7809e+0(8.22e−3)− 2.6802e+0(1.47e−2)− 3.4506e+0(4.65e−2)− 2.8071e+0(2.28e−3)− 2.5395e+0(4.28e−3) 10 4.0588e+0(2.07e−2)+ 6.0107e+0(8.38e−2)− 4.9239e+0(1.54e−1)− 4.4491e+0(3.28e−2)= 4.1394e+0(2.99e−2)+ 5.1763e+0(1.38e−1)− 4.5676e+0(1.29e−2)− 4.3855e+0(5.09e−2) WFG6 4 5.6918e−1(1.70e−2)= 7.0687e−1(4.75e−3)− 8.3477e−1(1.94e−2)− 7.2309e−1(1.68e−2)− 5.4759e−1(1.59e−2)+ 7.8510e−1(2.30e−2)− 5.4142e−1(4.45e−3)+ 5.7021e−1(1.90e−2) 6 1.4440e+0(1.21e−2)= 2.0196e+0(3.28e−2)− 2.0806e+0(3.74e−2)− 1.7276e+0(3.27e−2)− 1.5055e+0(1.59e−2)= 2.0403e+0(4.90e−2)− 1.4742e+0(1.19e−2)= 1.4330e+0(7.98e−2) 8 2.5793e+0(9.84e−3)+ 3.9350e+0(1.23e−1)− 3.5307e+0(3.85e−2)− 2.8925e+0(2.37e−2)− 2.7360e+0(2.54e−2)− 3.5280e+0(4.51e−2)− 2.8178e+0(2.15e−3)− 2.6016e+0(1.14e−3) 10 4.0973e+0(2.06e−2)= 6.6894e+0(3.79e−1)− 5.4594e+0(7.55e−2)− 4.5183e+0(6.74e−2)− 4.2177e+0(4.28e−2)− 5.3067e+0(1.06e−1)− 4.6155e+0(2.72e−2)− 4.0514e+0(6.64e−1) WFG7 4 5.7318e−1(4.76e−3)+ 5.3582e−1(5.05e−4)+ 6.7532e−1(9.57e−3)− 6.9963e−1(1.11e−2)− 5.2719e−1(1.70e−3)+ 6.6056e−1(1.73e−2)− 5.3778e−1(3.42e−3)+ 6.3809e−1(1.16e−2) 6 1.4365e+0(4.24e−3)+ 1.4876e+0(5.09e−4)= 1.5948e+0(3.68e−2)= 1.6996e+0(2.03e−2)− 1.4728e+0(1.73e−2)+ 1.6279e+0(3.79e−2)− 1.5057e+0(5.32e−3)= 1.5038e+0(1.95e−4) 8 2.5924e+0(9.00e−3)= 2.7408e+0(1.99e−2)− 2.6659e+0(1.73e−2)− 2.7574e+0(3.58e−2)− 2.6867e+0(1.72e−2)− 2.6904e+0(5.41e−2)− 2.8202e+0(4.20e−3)− 2.5487e+0(1.84e−2) 10 4.1210e+0(3.11e−2)− 4.3784e+0(3.01e−2)− 4.1397e+0(4.38e−2)− 4.3651e+0(3.00e−2)− 4.1492e+0(2.94e−2)− 4.3826e+0(6.58e−2)− 4.5659e+0(3.88e−2)− 4.0482e+0(4.54e−2) WFG8 4 5.9582e−1(3.33e−3)+ 5.7254e−1(1.85e−3)+ 6.5348e−1(1.03e−2)= 6.8670e−1(1.28e−2)− 6.4167e−1(1.57e−2)= 6.6406e−1(1.56e−2)− 5.7378e−1(2.42e−3)+ 6.4762e−1(5.79e−3) 6 1.5178e+0(7.53e−4)+ 1.4728e+0(1.15e−3)+ 1.5947e+0(6.81e−3)= 1.7163e+0(2.24e−2)− 1.6815e+0(9.32e−3)− 1.6320e+0(1.96e−2)− 1.4829e+0(7.02e−3)+ 1.6042e+0(1.74e−3) 8 2.7620e+0(6.66e−2)= 2.7623e+0(1.08e−2)= 2.8162e+0(1.05e−2)− 2.9715e+0(1.27e−2)− 3.1768e+0(1.11e−2)− 2.9208e+0(2.43e−2)− 2.8742e+0(4.42e−3)− 2.7587e+0(1.19e−2) 10 5.2260e+0(4.98e−2)− 4.3534e+0(4.50e−2)= 4.3977e+0(3.45e−2)− 4.5685e+0(3.94e−2)− 4.7632e+0(5.61e−2)− 4.3663e+0(2.29e−2)= 4.6152e+0(2.14e−2)− 4.3462e+0(1.26e−3) WFG9 4 5.5128e−1(8.97e−3)+ 5.2399e−1(7.70e−4)+ 6.0185e−1(1.37e−2)= 6.8666e−1(1.85e−2)− 5.2064e−1(7.62e−3)+ 6.2126e−1(1.74e−2)− 5.3495e−1(5.36e−3)+ 6.0844e−1(2.26e−2) 6 1.3999e+0(9.65e−3)= 1.4418e+0(4.45e−3)= 1.4949e+0(8.74e−3)− 1.6030e+0(2.14e−2)− 1.4574e+0(5.66e−3)= 1.5495e+0(1.01e−2)− 1.4296e+0(8.48e−3)= 1.4264e+0(2.32e−3) 8 2.5828e+0(5.78e−3)= 2.6762e+0(1.70e−2)− 2.6364e+0(1.38e−2)− 2.7980e+0(2.10e−2)− 2.7406e+0(1.62e−2)− 2.7461e+0(5.36e−2)− 2.7566e+0(4.21e−3)− 2.5674e+0(2.02e−2) 10 4.1484e+0(1.26e−3)− 4.2146e+0(8.66e−2)− 4.0020e+0(3.79e−2)− 4.3779e+0(4.39e−2)− 4.2196e+0(3.48e−2)− 4.2992e+0(5.40e−2)− 4.4885e+0(3.03e−2)− 3.9974e+0(1.96e−2) ± /= 9/14/13 5/25/6 0/30/6 1/31/4 7/21/8 0/33/3 8/22/6 Complex & Intelligent Systems (2021) 7:2697–2710 2707 Table 5 Comparison results of ANMPSO and seven MOEAs on WFG1–9 using HV Problem M GrEA RVEA NMPSO SPEA2-SDE Two-Arch2 SRA RPD-NSGAII ANMPSO WFG1 4 9.5191e−1(4.73e−3)− 4.2183e−1(5.91e−2)− 6.1561e−1(2.51e−2)− 9.8607e−1(1.14e−3)+ 9.8907e−1(6.49e−4)+ 3.6914e−1(5.44e−2)− 9.8748e−1(1.48e−3)+ 9.7211e−1(2.42e−2) 6 9.8582e−1(3.41e−3)+ 3.6622e−1(6.79e−2)− 2.8961e−1(2.88e−2)− 9.9670e−1(7.08e−4)+ 9.6377e−1(3.29e−2)+ 3.0001e−1(3.00e−2)− 9.9082e−1(6.04e−3)− 9.4577e−1(2.65e−2) 8 9.9128e−1(8.56e−4)+ 3.2439e−1(2.22e−2)− 2.5749e−1(3.62e−2)− 9.9723e−1(1.58e−3)+ 7.8205e−1(2.01e−2)− 2.4996e−1(3.00e−3)− 9.1831e−1(6.68e−2)− 9.2161e−1(4.31e−2) 10 9.8215e−(2.35e−3)= 3.9139e−1(4.98e−2)− 3.1395e−1(5.68e−2)− 9.7756e−1(5.13e−4)= 8.3941e−1(7.54e−2)− 2.5300e−1(2.91e−2)− 9.7478e−1(6.05e−3)= 9.7989e−1(3.72e−2) WFG2 4 9.6235e−1(6.19e−3)− 9.3720e−1(1.21e−2)− 9.8254e−1(3.91e−3)= 9.7610e−1(4.54e−3)− 9.8189e−1(5.09e−4)= 9.8597e−1(8.01e−2)= 9.8710e−1(1.13e−3)= 9.8799e−1(6.66e−2) 6 9.7214e−1(2.25e−3)= 9.8643e−1(1.91e−2)+ 9.4307e−1(5.08e−3)− 9.8898e−1(1.61e−3)+ 9.9550e−1(2.56e−4)+ 9.7743e−1(1.57e−2)+ 9.9334e−1(1.61e−3)+ 9.7310e−1(8.09e−2) 8 9.8484e−1(3.79e−3)− 8.8722e−1(1.68e−2)− 9.4909e−1(7.52e−3)− 9.8993e−1(1.15e−3)= 9.9210e−1(1.96e−3)= 9.4870e−1(4.00e−3)− 9.8969e−1(2.07e−3)− 9.9322e−1(1.16e−2) 10 9.6889e−1(1.51e−3)= 8.9271e−1(3.97e−2)− 9.6670e−1(8.19e−2)= 9.9033e−1(1.54e−3)+ 9.9498e−1(1.04e−3)= 9.3715e−1(1.27e−2)− 9.8912e−1(2.25e−3)− 9.7147e−1(8.15e−2) WFG3 4 2.4768e−1(5.22e−3)− 2.0495e−1(2.38e−2)− 2.3258e−1(9.30e−3)= 2.3221e−1(1.83e−2)= 2.9040e−1(5.29e−3)= 1.1481e−1(2.68e−2)− 2.6247e−1(5.76e−3)− 2.9210e−1(1.73e−2) 6 1.5853e−1(1.56e−2)− 1.0546e−2(1.85e−2)− 1.0884e−2(8.57e−3)− 4.6669e−2(3.76e−2)− 1.0711e−1(3.00e−2)+ 0.0000e+0(0.00e+0)− 1.2294e−1(1.13e−2)− 2.5949e−1(2.07e−2) 8 1.5835e−2(1.87e−2)− 0.0000e+0(0.00e+0)− 0.0000e+0(0.00e+0)− 4.0371e−3(8.07e−3)− 6.9561e−3(7.86e−3)− 0.0000e+0(0.00e+0)= 4.3325e−2(9.41e−3)+ 2.0265e−2(4.05e−3) 10 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0)= 0.0000e+0(0.00e+0) WFG4 4 7.0883e−1(1.30e−3)− 6.3529e−1(6.71e−3)− 6.5878e−1(8.65e−4)− 6.9501e−1(2.84e−3)− 6.9257e−1(1.11e−3)− 5.9825e−1(1.23e−2)− 7.1018e−1(4.36e−4)− 7.3046e−1(3.68e−3) 6 8.3899e−1(2.51e−3)+ 7.9805e−1(1.09e−2)+ 7.7198e−1(7.08e−3)− 8.3046e−1(4.41e−3)+ 7.7711e−1(2.68e−3)− 6.7792e−1(6.52e−3)− 8.6524e−1(1.42e−3)+ 7.8684e−1(2.45e−2) 8 8.4915e−1(3.11e−3)− 7.5362e−1(7.87e−3)− 8.1360e−1(1.15e−2)− 8.6283e−1(2.69e−3)= 8.1790e−1(7.89e−3)− 6.8234e−1(2.10e−2)− 9.2870e−1(1.90e−3)+ 8.6110e−1(2.93e−2) 10 9.3539e−1(5.82e−3)− 7.4836e−1(4.36e−2)− 8.2715e−1(8.99e−3)− 8.9669e−1(6.90e−4)− 7.9367e−1(6.54e−3)− 6.6739e−1(2.31e−2)− 9.5088e−1(1.31e−3)− 9.7224e−1(4.41e−2) WFG5 4 6.6462e−1(2.37e−4)= 6.1794e−1(1.82e−3)− 6.3523e−1(3.60e−3)− 6.5625e−1(2.71e−3)− 6.4921e−1(3.54e−3)− 5.7225e−1(1.01e−2)− 6.6553e−1(2.02e−3)− 6.7792e−1(2.21e−3) 6 8.0106e−1(2.52e−3)− 7.0636e−1(2.15e−2)− 7.5054e−1(2.67e−3)− 7.8669e−1(2.21e−3)− 7.4542e−1(2.25e−3)− 6.2720e−1(1.42e−2)− 8.1719e−1(5.66e−4)− 8.3211e−1(2.55e−2) 8 8.1111e−1(5.00e−3)+ 7.2160e−1(3.58e−2)− 7.9280e−1(5.29e−3)+ 8.3210e−1(4.55e−3)+ 7.7536e−1(5.85e−3)+ 6.2934e−1(2.33e−2)− 8.7983e−1(2.10e−4)+ 7.3110e−1(1.84e−2) 10 8.9103e−1(2.03e−3)= 7.7689e−1(5.52e−3)− 8.0962e−1(6.37e−3)− 8.4931e−1(3.06e−3)− 7.4392e−1(8.92e−3)− 6.3544e−1(4.25e−2)− 8.9780e−1(2.67e−4)= 8.9607e−1(3.26e−2) WFG6 4 6.4411e−1(1.89e−2)− 6.0850e−1(1.32e−2)− 5.3704e−1(1.60e−3)− 6.4238e−1(1.07e−2)− 6.5088e−1(2.78e−2)= 6.5144e−1(2.73e−2)= 6.5073e−1(3.69e−3)= 6.5487e−1(1.21e−2) 6 7.8329e−1(8.62e−3)− 6.3522e−1(1.56e−2)− 6.5403e−1(2.87e−3)− 7.6451e−1(1.43e−2)= 7.4056e−1(1.33e−2)− 7.7590e−1(1.42e−2)+ 7.8670e−1(3.52e−2)+ 7.7428e−1(1.44e−2) 8 7.7688e−1(7.70e−3)− 6.0895e−1(7.00e−2)− 7.0901e−1(1.57e−3)− 8.1846e−1(1.40e−2)− 7.3845e−1(1.25e−2)− 5.6990e−1(1.88e−2)− 8.5389e−1(2.63e−2)− 8.6617e−1(9.60e−3) 10 8.7303e−1(1.62e−2)= 6.1924e−1(1.01e−1)− 8.2579e−1(4.12e−3)− 8.4356e−1(1.61e−2)− 7.2085e−1(2.22e−2)− 5.7541e−1(2.52e−2)− 8.6942e−1(2.40e−2)= 8.7359e−1(1.75e−2) WFG7 4 7.1282e−1(1.22e−3)= 7.0764e−1(2.02e−4)= 7.0953e−1(1.32e−4)= 7.0236e−1(1.67e−3)− 6.9649e−1(1.43e−3)− 6.8426e−1(2.52e−3)− 7.1186e−1(8.30e−4)= 7.1340e−1(3.33e−3) 6 8.6251e−1(1.95e−3)= 8.6336e−1(1.07e−3)= 8.6017e−1(1.33e−3)= 8.4205e−1(1.60e−3)− 8.1521e−1(3.31e−3)− 8.6695e−1(6.58e−3)= 8.6972e−1(5.99e−4)= 8.6845e−1(5.40e−3) 8 8.6795e−1(2.88e−3)− 9.2059e−1(9.92e−4)+ 9.1728e−1(2.02e−3)+ 9.0380e−1(5.60e−3)+ 8.4840e−1(3.48e−3)− 8.7026e−1(6.52e−3)− 9.3790e−1(7.30e−4)+ 8.9953e−1(1.72e−3) 10 9.5603e−1(2.30e−3)= 9.3315e−1(6.57e−3)− 9.4154e−1(2.86e−3)− 9.3087e−1(3.61e−3)− 8.1378e−1(4.87e−3)− 8.5974e−1(1.19e−2)− 9.5798e−1(2.08e−3)= 9.5977e−1(8.18e−3) WFG8 4 6.1905e−1(6.63e−4)= 6.1239e−1(2.88e−3)− 6.1025e−1(4.14e−4)− 6.1271e−1(1.48e−3)− 5.9536e−1(6.35e−4)− 5.7617e−1(7.60e−3)− 6.0831e−1(9.62e−4)− 6.2029e−1(5.33e−3) 6 7.2055e−1(3.22e−3)− 7.3297e−1(6.51e−3)− 7.4772e−1(2.34e−3)− 7.4257e−1(5.66e−3)− 6.6315e−1(8.91e−3)− 6.8459e−1(8.27e−3)− 7.5289e−1(2.22e−3)= 7.5108e−1(2.11e−3) 8 6.9532e−1(1.96e−2)− 8.4904e−1(5.61e−2)− 8.1153e−1(3.94e−3)− 8.1084e−1(4.72e−3)− 6.5025e−1(7.94e−3)− 7.2738e−1(1.38e−2)− 8.3090e−1(1.49e−3)− 8.6011e−1(5.73e−2) 10 8.5422e−1(3.08e−3)− 6.8893e−1(7.02e−2)− 8.6352e−1(5.69e−3)− 8.4624e−1(6.60e−3)− 5.7785e−1(3.15e−2)− 8.7995e−1(1.36e−2)= 8.7377e−1(8.41e−3)− 8.8454e−1(1.48e−2) WFG9 4 6.7475e−1(1.02e−3)− 6.7888e−1(3.70e−4)− 6.8437e−1(2.04e−3)= 6.7007e−1(1.38e−3)− 6.6617e−1(2.19e−3)− 6.5781e−1(2.23e−3)− 6.6105e−1(3.90e−3)− 6.8652e−1(3.79e−3) 6 8.0717e−1(2.93e−3)= 7.9970e−1(8.41e−3)= 8.1429e−1(4.67e−3)+ 7.9671e−1(4.25e−3)= 7.6923e−1(3.37e−3)− 7.7841e−1(6.68e−3)− 8.1420e−1(5.15e−3)+ 8.0278e−1(4.75e−3) 8 8.1448e−1(2.68e−3)− 8.4035e−1(1.21e−2)− 8.5510e−1(9.40e−3)− 8.4140e−1(4.79e−3)− 7.8065e−1(1.66e−2)− 8.1642e−1(6.13e−3)− 8.7446e−1(1.05e−3)= 8.7627e−1(2.75e−3) 10 8.4106e−1(2.33e−3)− 8.5809e−1(4.10e−3)− 8.5523e−1(6.05e−3)− 8.5061e−1(4.11e−3)− 7.3924e−1(1.32e−2)− 8.3151e−1(3.66e−3)− 8.7976e−1(5.84e−3)− 8.8504e−1(5.58e−3) ± /= 4/20/12 3/29/4 3/26/7 8/21/7 5/25/6 2/28/6 9/16/11 Complex & Intelligent Systems (2021) 7:2697–2710 2708 Fig. Comparisons results on WFG1–WFG9 2 Solution set correspond- ing to the best IGD values for all algorithms on WFG3 prob- lems with 10 objectives Fig. 3 Solution set correspond- ing to the best IGD values for all algorithms on WFG5 prob- lems with 10 objectives Fig. 2 Solution set correspond- ing to the best IGD values for all algorithms on WFG3 prob- lems with 10 objectives Fig. 2 Solution set correspond- ing to the best IGD values for all algorithms on WFG3 prob- lems with 10 objectives Fig. 3 Solution set correspond- ing to the best IGD values for all algorithms on WFG5 prob- lems with 10 objectives “offset-frontal crash”. The nature of the Pareto-optimal Front is complex with holes and the density of its points is variable. “offset-frontal crash”. The nature of the Pareto-optimal Front is complex with holes and the density of its points is variable. cover the whole true PF with a competitive diversity. ANMPSO is better than SPEA2-SDE, Two-Arch2, SRA, and RPD-NSGAII. Besides, RVEA is relatively better than ANMPSO, since it is more evenly distributed. As observed from Fig. 3, the result of ANMPSO also shows an excel- lent performance with covering the true PF entirely. This uniform distribution is credit to parameter adjustments and the guidance of gbest. The local exploitation ability and global exploration ability are improved with adaptive strategy. cover the whole true PF with a competitive diversity. ANMPSO is better than SPEA2-SDE, Two-Arch2, SRA, and RPD-NSGAII. Besides, RVEA is relatively better than ANMPSO, since it is more evenly distributed. As observed from Fig. 3, the result of ANMPSO also shows an excel- lent performance with covering the true PF entirely. This uniform distribution is credit to parameter adjustments and the guidance of gbest. The local exploitation ability and global exploration ability are improved with adaptive strategy. As such, the multi-objective optimization problem is for- mulated as (18) min F(퐱) = [Mass, Ain, Intrusion] s.t. 1 mm ≤퐱≤3 mm, (18) Table 6 IGD and HV values of all the algorithms on the crash safety design of vehicles problem The better average values of the three algorithms are bold Algorithms IGD HV ANMPSO 0.3833 0.0367 GrEA 0.4503 0.0365 RVEA 1.0120 0.0337 NMPSO 2.2550 0.0333 SPEA2-SDE 0.6039 0.0351 Two-Arch2 0.3571 0.0381 SRA 1.0241 0.0349 RPD-NSGAII 0.3697 0.0378 Table 6 IGD and HV values of all the algorithms on the crash safety design of vehicles problem Application to the crash safety design of vehicles To verify the availability and practicability of the proposed ANMPSO algorithm, it is applied to optimize the problem of the crash safety design of vehicles. This problem aims to optimize the crash-worthiness of vehicle frontal struc- ture. Considering the lightweight, the mass of the vehicle is set as the first objective. The second objective is to select an integration of collision acceleration in the “full frontal crash”. Considering the most severe mechanical damage, the third objective is set as the toe board intrusion in the 1 3 Complex & Intelligent Systems (2021) 7:2697–2710 2709 where x = (t1, t2, t3, t4, t5)T, Ain = ∫t2 t1 adt . The detailed information can be found in [2]. where x = (t1, t2, t3, t4, t5)T, Ain = ∫t2 t1 adt . The detailed information can be found in [2]. Although ANMPSO performs well in MaOPs, it has some drawbacks in solving multi-modal problems. In addition, the performance of ANMPSO on other application problems with more objectives in the real world needs to be studied in the future. where x = (t1, t2, t3, t4, t5)T, Ain = ∫t2 t1 adt . The detailed information can be found in [2]. For this problem, all objectives are to be minimized. The maximum number of iterations is set to 1000 and the population size is set to 100. In this problem, the number of decision variables D is 5. To make a fair comparison, all the experiments are run 10 times with different random seeds, and the average IGD and HV values in 10 runs are collected for comparison. The comparison results are pre- sented in Table 6. Acknowledgements This work was supported by the National Natural Science Foundation of China under Grant no. 51774228 and Grant no. 52074205, in part by Shaanxi province fund for Distinguished Young Scholars under Grant no. 2020JC-44, in part by Joint Fund Project of Shaanxi Province Natural Science Basic Research Program under Grant no. 2019 JLP-16. From Table 6, it can be observed that ANMPSO, Two- Arch2 and RPD-NSGAII achieve competitive performances. The results of Two-Arch2 and RPD-NSGAII are slightly better than ANMPSO. RVEA, NMPSO, and SRA are signifi- cantly worse than ANMPSO, because they do not even find non-dominated solutions. This poor performance is mainly due to the favor of poorly converged individuals in regions where the objective space is less crowded. Conclusions In this paper, ANMPSO is proposed to balance convergence and diversity which has a competitive performance. It can effectively select gbest and obtain a better distribution in the searching space. The proposed algorithm can prevent particles from trapping into local optima. Numerical experi- mental results prove that the proposed ANMPSO can obtain excellent performances in the two sets of MaOP benchmark functions and the practical crash safety design of vehicles. Declarations Conflict of interest The authors declare that there are no conflicts of interest regarding the publication of this paper, and the comparison results data used to support the findings of this study are available from the corresponding author upon request. 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Application to the crash safety design of vehicles The reason why ANMPSO outperformed these algorithms is that it is suit- able for tackling this problem with disconnect PF. The spe- cial gbest selection mechanism can guide particles close to the true PF. 1 3 References Environ Monit Assess 192:767–794 pp 9. Zhang Q, Li H (2007) MOEA/D: a multiobjective evolutionary algorithm based on decomposition. IEEE Trans Evol Comput 11:712–731 29. Li L, Li G, Chang L (2020) A many-objective particle swarm optimization with grid dominance ranking and clustering. Appl Soft Comput 96:1–11 10. Elarbi M, Bechikh S, Gupta A, Said LB, Ong YS (2018) A new decomposition-based NSGA-II for many-objective optimization. IEEE Trans Syst Man Cybern B 48:1191–1210 p y j IEEE Trans Syst Man Cybern B 48:1191–1210 30. Chaman Garcia I, Coello Coello CA, Arias-Montano (2014) MOPSOhv: a new hypervolume-based multi-objective particle swarm optimizer. 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Praditwong K, Harman M, Yao X (2011) Software module cluster- ing as a multi-objective search problem. IEEE Trans Softw Eng 37:264–282 Some advantages of ANMPSO can be summarized from the experimental results: (1) By analyzing the comparison results, the comparisons between ANMPSO and GrEA, RVEA, NMPSO, SPEA2-SDE, Two-Arch2, SRA, and RPD- NSGAII evident that ANMPSO obtains the best IGD values and HV values, especially in the DTLZ4, DTLZ7, WFG3, and WFG5 test functions. However, from the HV compari- son results on DTLZ1 and DTLZ3 problems, ANMPSO cannot effectively tackle the multi-modal problem. (2) By comparing with the curves of IGD metric values on the DTLZ4 and DTLZ7 with 10 objectives, it can be seen that ANMPSO has higher convergence speed and precision dur- ing the whole iteration process. (3) By comparing with the non-dominance solution of these compared algorithms, it can be observed that ANMPSO can fully cover the true PF. 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https://openalex.org/W2924553683	https://www.biorxiv.org/content/biorxiv/early/2019/03/27/591271.full.pdf	English	null	Bioactivity-guided isolation of rosmarinic acid as a principle bioactive compound from the butanol extract of <i>Isodon rugosus</i> against pea aphid, <i>Acyrthosiphon pisum</i>	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	13,066	. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 3 Acyrthosiphon pisum 4 Saira Khan1,2,3,4, Clauvis Nji Tizi Taning2, Elias Bonneure3, Sven Mangelinckx3, Guy Smagghe2, Raza 5 Ahmad1, Nighat Fatima5, Muhammad Asif6, Mohammad Maroof Shah1* 6 7 1Biotechnology Program, Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad 8 Campus, Pakistan 9 2Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium 10 3Department of Green Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, 11 Belgium 12 4Current address: Department of Biotechnology, Hazara University Mansehra, Mansehra, Pakistan 13 5Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Pakistan 14 6Department of Management Sciences, COMSATS University Islamabad, Abbottabad Campus, Pakistan 15 16 *Corresponding Author 17 E-mail: mmshah@cuiatd.edu.pk (MS) 1 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 18 Abstract 19 Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest 20 control involving the excessive use of synthetic insecticides over decades has led to multiple forms of aphid 21 resistance to most classes of insecticides. In nature, plants produce secondary metabolites during their interaction 22 with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and deterrents towards 23 the insects. In a previous study, we demonstrated that the butanol fraction from a crude methanolic extract of an 24 important plant species, Isodon rugosus showed strong insecticidal activity against the pea aphid, Acyrthosiphon 25 pisum. It was however not known as which compound was responsible for such activity. To further explore this 26 finding, current study aimed to exploit a bioactivity-guided strategy to isolate and identify the active compound in 27 the butanol fraction of I. rugosus. As such, reversed-phase flash chromatography, acidic extraction and different 28 spectroscopic techniques were used to isolate and identify the new compound, rosmarinic acid as the bioactive 29 compound in I. rugosus. Insecticidal activity of rosmarinic acid was carried out using standard protocols on A. 30 pisum. The data was analyzed using qualitative and quantitative statistical approaches. Considering that a very low 31 concentration of this compound (LC90 = 5.4 ppm) causes significant mortality in A. pisum within 24 h, rosmarinic 32 acid could be exploited as a potent insecticide against this important pest insect. Furthermore, I. rugosus is already 33 used for medicinal purposes and rosmarinic acid is known to reduce genotoxic effects induced by chemicals, hence 34 it is expected to be safer compared to the current conventional pesticides. While this study highlights the potential of 35 I. rugosus as a possible biopesticide source against A. pisum, it also provides the basis for further exploration and 36 development of formulations for effective field application. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 37 Introduction 2 38 Aphids are among the most important agricultural pest insects of many crops worldwide. They feed exclusively on 39 plant phloem sap by inserting their needle-shaped mouthparts into sieve elements, usually resulting to the stunting, 40 discoloration and deformation of plants, while the growth of sooty molds on honeydew produced by these insects 41 reduces the economic value of crops [1, 2]. Moreover, aphids are also vectors of many important plant viruses [3-5]. 42 The pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae), adversely affects economically important legume 43 crops worldwide. It is oligophagous, comprising of a number of biotypes or races living on a number of legume 44 hosts (red clover, pea and broad bean and alfalfa races) [6-9]. Current aphid control strategies predominantly rely on 2 38 Aphids are among the most important agricultural pest insects of many crops worldwide. They feed exclusively on 39 plant phloem sap by inserting their needle-shaped mouthparts into sieve elements, usually resulting to the stunting, 40 discoloration and deformation of plants, while the growth of sooty molds on honeydew produced by these insects 41 reduces the economic value of crops [1, 2]. Moreover, aphids are also vectors of many important plant viruses [3-5]. 42 The pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae), adversely affects economically important legume 43 crops worldwide. It is oligophagous, comprising of a number of biotypes or races living on a number of legume 44 hosts (red clover, pea and broad bean and alfalfa races) [6-9]. Current aphid control strategies predominantly rely on 2 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 45 the use of insecticides such as carbamates, organophosphates, pyrethroids, neonicotinoids and pymetrozine [10]. 37 Introduction 46 However, the frequent use of these insecticides over the decades has led to multiple forms of aphid resistance to 47 most classes of insecticides, making it very difficult to control this insect pest [11]. 47 most classes of insecticides, making it very difficult to control this insect pest [11]. 48 The use of botanical pesticides could present a safe alternative compared to the use of broad spectrum 49 chemical insecticides in crop protection [12, 13]. In nature, plants produce secondary metabolites during their 50 interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and 51 deterrents towards the insects [14-16]. Because of such wide insecticidal properties, the study of secondary 52 metabolites and the development of new potent formulations based on them has become increasingly important. 53 Screening of plant extracts followed by bioactivity-guided fractionation, isolation and identification of active 54 principles is considered to be one of the most successful strategies for the discovery of bioactive natural products 55 against insect pests [17]. 56 Isodon rugosus (Wall. ex Benth.) Codd (syn. Plectranthus rugosus Wall. ex. Benth.) is an aromatic 57 branched shrub, belonging to the family Lamiaceae. The plant is used in Pakistani traditional medicine for many 58 diseases as an antiseptic, hypoglycemic, antidiarrheal and as bronchodilator [18, 19]. Among many other traditional 59 medicinal uses, the plant extracts and different solvent fractions are known to be effective as antifungal, 60 antibacterial, phytotoxic and antioxidant agents and are able to show lipoxygenase inhibitory activities [20-23]. 61 Based on phytochemical studies, this plant is known to contain steroids, flavonoids, terpenoids, saponins, tannins, 62 cardiac glycosides, coumarins, reducing sugars and β-cyanin. Diterpenoids (rugosinin, effusanin-A, effusanin-B, 63 effusanin-E, lasiokaurin and oridonin) and triterpenoids (plectranthoic acid A and B, acetyl plectranthoic acid and 64 plectranthadiol) have also been successfully isolated from this plant [24-26]. However, despite several studies on the 65 bioactivity of I. rugosus where most efforts were focused towards human health, none of these have isolated and 66 assessed the insecticidal activity of compounds from this plant. In a previous study, we evaluated the aphicidal 67 properties of the hexane, dichloromethane, butanol and ethyl acetate fractions of a crude methanolic extract from I. 68 rugosus, and confirmed that the butanol fraction showed the best activity against the pea aphid, A. pisum [27]. To 69 further explore this finding, a bioactivity-guided strategy against A. 72 Insects 73 A continuous colony of A. pisum was maintained on faba bean plants (Vicia faba) in the Laboratory of Agrozoology 74 at Ghent University, Belgium at 23–25 °C and 65±5% relative humidity (RH) under a 16:8 h light: dark photoperiod 75 [28]. All the bioassays were performed under these conditions. Newly born nymphs (˂ 24 h old) of A. pisum were 76 used for all the bioassays. By gentle probing of the aphids with a brush and also by observing post-mortem color 77 change of the body, mortality was assessed after 24 h of treatment. 73 A continuous colony of A. pisum was maintained on faba bean plants (Vicia faba) in the Laboratory of Agrozoology 74 at Ghent University, Belgium at 23–25 °C and 65±5% relative humidity (RH) under a 16:8 h light: dark photoperiod 75 [28]. All the bioassays were performed under these conditions. Newly born nymphs (˂ 24 h old) of A. pisum were 76 used for all the bioassays. By gentle probing of the aphids with a brush and also by observing post-mortem color 77 change of the body, mortality was assessed after 24 h of treatment. 78 Plant collection and extraction 79 The aerial parts of I. rugosus were collected from lower Northern areas of Pakistan in the month of October, 2012. 80 The plant material was shade-dried for up to 3 months and ground to powder using an electric grinder. 1 kg of the 81 dried powder was soaked in a glass jar containing 3 L of methanol at room temperature. After two days, the solvent 82 layer was filtered with Whatman filter paper No. 1 and this process was repeated three times. The resulting filtrate 83 was concentrated by using a rotary evaporator at 35 °C and the obtained crude methanolic extract was stored at 4 °C 84 [29, 27]. For fractionation, 90 g dried crude methanolic extract was mixed with five parts of water and then 85 extracted successively by n-hexane (4 × 150 mL), dichloromethane (4 × 150 mL), ethyl acetate (4 ×150 mL) and n- 86 butanol (4 × 150 mL) as described by Khan et al. [27]. All the fractions were concentrated using a rotary evaporator 87 under reduced pressure at 40 °C. The resulting extracts were stored in a refrigerator at 4 °C until further use. 37 Introduction CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 37 Introduction pisum was used to isolate and identify the active 70 compound in the butanol fraction of I. rugosus. 71 Materials and methods 48 The use of botanical pesticides could present a safe alternative compared to the use of broad spectrum 49 chemical insecticides in crop protection [12, 13]. In nature, plants produce secondary metabolites during their 50 interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and 51 deterrents towards the insects [14-16]. Because of such wide insecticidal properties, the study of secondary 52 metabolites and the development of new potent formulations based on them has become increasingly important. 53 Screening of plant extracts followed by bioactivity-guided fractionation, isolation and identification of active 54 principles is considered to be one of the most successful strategies for the discovery of bioactive natural products 55 against insect pests [17]. 56 Isodon rugosus (Wall. ex Benth.) Codd (syn. Plectranthus rugosus Wall. ex. Benth.) is an aromatic 57 branched shrub, belonging to the family Lamiaceae. The plant is used in Pakistani traditional medicine for many 58 diseases as an antiseptic, hypoglycemic, antidiarrheal and as bronchodilator [18, 19]. Among many other traditional 59 medicinal uses, the plant extracts and different solvent fractions are known to be effective as antifungal, 60 antibacterial, phytotoxic and antioxidant agents and are able to show lipoxygenase inhibitory activities [20-23]. 61 Based on phytochemical studies, this plant is known to contain steroids, flavonoids, terpenoids, saponins, tannins, 62 cardiac glycosides, coumarins, reducing sugars and β-cyanin. Diterpenoids (rugosinin, effusanin-A, effusanin-B, 63 effusanin-E, lasiokaurin and oridonin) and triterpenoids (plectranthoic acid A and B, acetyl plectranthoic acid and 64 plectranthadiol) have also been successfully isolated from this plant [24-26]. However, despite several studies on the 65 bioactivity of I. rugosus where most efforts were focused towards human health, none of these have isolated and 66 assessed the insecticidal activity of compounds from this plant. In a previous study, we evaluated the aphicidal 67 properties of the hexane, dichloromethane, butanol and ethyl acetate fractions of a crude methanolic extract from I. 68 rugosus, and confirmed that the butanol fraction showed the best activity against the pea aphid, A. pisum [27]. To 69 further explore this finding, a bioactivity-guided strategy against A. pisum was used to isolate and identify the active 70 compound in the butanol fraction of I. rugosus. 3 . . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 88 Isolation of the bioactive principle 89 Based on bioassays conducted by Khan et al, [27] the butanol extract presented the best biological activity against A. 90 pisum and was hence selected in this study for further bioactivity-guided fractionation and identification of the 91 active principle. The butanol extract (500 mg) was eluted with a Reveleris automated flash chromatography 92 instrument on a 12 g C18 pre-packed column (GRACE, Columbia, MD, US) starting with 100% water. The gradient 93 was ramped to 100% methanol over 60 column volumes (CV) and after collection of 95 fractions, the solid phase 94 was flushed with 5 CV acetonitrile. The flow rate was set to 30 mL/min (Table 1). Based on the UV spectral data, 95 the 95 fractions were combined into a total of 14 subfractions. These combined fractions were evaporated under 96 reduced pressure at 45 °C and finally under a high vacuum resulting in 14 subfractions (1A- 14A) (Table 2). The 14 97 subfractions were evaluated for their bioactivity against A. pisum, of which fraction 3A was selected on the basis of 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 98 maximum bioactivity for further fractionation through preparative liquid chromatography (prep-LC). A 10% 99 solution of fraction 3A was prepared in methanol. Two solvents were used, water (solvent A) and acetonitrile 100 (solvent B). A gradient was set starting with 100% solvent A from 0 to 100 min. From 100 min to 110 min, solvent 101 B went from 18% to 100% and stayed at 100% until 128 min, and then to 0% at 128.10 min and stayed at 0% until 102 132.10 min. After concentration under reduced pressure with a rotary evaporator and finally under a high vacuum, 103 three fractions, 3A-1, 3A-2 and 3A-3 were obtained. 88 Isolation of the bioactive principle Fraction 3A-3 was selected for active compound identification 104 (NMR and LC-MS) on the basis of the bioactivity against A. pisum. This compound was obtained in pure form by 105 doing a second flash chromatographic separation of 5 g of butanol extract and by using the run conditions as 106 mentioned in Table 3. From the second flash chromatography, a total of 354 fractions were collected which were 107 combined into six fractions, 1B, 2B, 3B, 4B, 5B and 6B on the basis of UV spectra and were further analyzed for 108 their bioactivity after concentration with a rotary evaporator under reduced pressure and high vacuum (Table 4). 109 Fraction 1B was selected for further purification on the basis of best bioactivity. On the basis of knowledge 110 regarding the acidic compound present in sub fraction 3A-3 (from 1H NMR and HPLC-MS analysis), an extraction 111 under acidic conditions was done to isolate the active compound from sub fraction 1B. For this purpose, 200 mg of 112 fraction 1B was dissolved in 10 mL of distilled water and acidified with 4 drops of hydrochloric acid (12 M). 113 Following extraction with ethyl acetate (four times 5 mL), two phases, ethyl acetate and aqueous, were obtained. 114 Both the ethyl acetate and the aqueous phase were concentrated. The ethyl acetate phase fraction was more bioactive 115 with lower LC values. Last traces of ethyl acetate were removed azeotropically with toluene and evaporation under 116 high vacuum of the residues resulted in 60 mg from the ethyl acetate phase and 60 mg from the aqueous phase. The 117 purified active principle was identified through different spectroscopic techniques. 98 maximum bioactivity for further fractionation through preparative liquid chromatography (prep-LC). A 10% 99 solution of fraction 3A was prepared in methanol. Two solvents were used, water (solvent A) and acetonitrile 100 (solvent B). A gradient was set starting with 100% solvent A from 0 to 100 min. From 100 min to 110 min, solvent 101 B went from 18% to 100% and stayed at 100% until 128 min, and then to 0% at 128.10 min and stayed at 0% until 102 132.10 min. After concentration under reduced pressure with a rotary evaporator and finally under a high vacuum, 103 three fractions, 3A-1, 3A-2 and 3A-3 were obtained. 88 Isolation of the bioactive principle Fraction 3A-3 was selected for active compound identification 104 (NMR and LC-MS) on the basis of the bioactivity against A. pisum. This compound was obtained in pure form by 105 doing a second flash chromatographic separation of 5 g of butanol extract and by using the run conditions as 106 mentioned in Table 3. From the second flash chromatography, a total of 354 fractions were collected which were 107 combined into six fractions, 1B, 2B, 3B, 4B, 5B and 6B on the basis of UV spectra and were further analyzed for 108 their bioactivity after concentration with a rotary evaporator under reduced pressure and high vacuum (Table 4). 109 Fraction 1B was selected for further purification on the basis of best bioactivity. On the basis of knowledge 110 regarding the acidic compound present in sub fraction 3A-3 (from 1H NMR and HPLC-MS analysis), an extraction 111 under acidic conditions was done to isolate the active compound from sub fraction 1B. For this purpose, 200 mg of 112 fraction 1B was dissolved in 10 mL of distilled water and acidified with 4 drops of hydrochloric acid (12 M). 113 Following extraction with ethyl acetate (four times 5 mL), two phases, ethyl acetate and aqueous, were obtained. 114 Both the ethyl acetate and the aqueous phase were concentrated. The ethyl acetate phase fraction was more bioactive 115 with lower LC values. Last traces of ethyl acetate were removed azeotropically with toluene and evaporation under 116 high vacuum of the residues resulted in 60 mg from the ethyl acetate phase and 60 mg from the aqueous phase. The 117 purified active principle was identified through different spectroscopic techniques. 5 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint Table 1. First reversed-phase flash chromatography conditions of butanol fraction (500 mg) from Isodon rugosus Run Conditions Cartridge Reveleris 12 g C18 40 µm Solvent A Water Solvent B Methanol Solvent C Acetonitrile Flow rate 30 mL/min Injection type Dry sample ELSD Carrier Isopropanol Per vial volume 25 mL UV1 Wavelength 220 nm UV2 Wavelength 254 nm Table 1. 88 Isolation of the bioactive principle First reversed-phase flash chromatography conditions of butanol fraction (500 mg) from Isodon rugosus reversed-phase flash chromatography conditions of butanol fraction (500 mg) from Isodon 118 119 rugosus 6 6 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 121 Table 2. Subfractions (1A-14A) collected from the first reversed-phase flash chromatography of butanol 122 extract (500 mg) Fractions Weight (mg) 1A 52 2A 11 3A 46 4A 7 5A 20 6A 15 7A 54 8A 58 9A 14 10A 49 11A 18 12A 15 13A 21 14A 18 21 Table 2. Subfractions (1A-14A) collected from the first reversed-phase flash chromatography of butanol 22 extract (500 mg) actions (1A-14A) collected from the first reversed-phase flash chromatography of butano 121 122 7 7 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 124 Table 3. 88 Isolation of the bioactive principle Second reversed-phase flash chromatography conditions of butanol fraction (5 g) of Isodon rugosus Run Conditions Cartridge Reveleris 120 g C18 40 µm Solvent A Water Solvent B Methanol Flow rate 85 mL/min Injection type Dry sample ELSD Carrier Isopropanol Per vial volume 25 mL UV1 Wavelength 220 nm UV2 Wavelength 254 nm 125 124 nd reversed-phase flash chromatography conditions of butanol fraction (5 g) of Isodon rugosu 126 Table 4. Subfractions (1B-6B) from the second reversed-phase flash chromatography of butanol extract (5 g) Fractions Weight (mg) 1B 530 2B 830 3B 1523 4B 195 5B 140 6B 128 127 126 Table 4. Subfractions (1B-6B) from the second reversed-phase flash chromatography of butanol extract (5 g) 127 128 Identification of the bioactive compound 129 Mass spectra were recorded using a HPLC-MS instrument consisting of an Agilent (Waldbronn, Germany) model 130 1100 liquid chromatograph with a diode array detector coupled with a mass spectrometer with electrospray 131 ionization geometry (Agilent MSD 1100 series). The prep-LC consisted of an Agilent 1100 Series liquid 132 chromatograph using a Supelco Ascentis C18 column (I.D. x L 21.2 mm x 150 mm, 5 µm particle size) connected to 8 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 133 an UV-VIS variable wavelength detector (VWD) and automatic fraction collector. Flash chromatography was 134 performed with the Reveleris Flash System (GRACE). 1H and 13C NMR spectra were obtained on a BRUKER 135 Advance III 400 spectrometer. All the solvents and chemicals used were of analytical grade. Optical rotation was 136 taken with a JASCO P-2000 series polarimeter. 133 an UV-VIS variable wavelength detector (VWD) and automatic fraction collector. Flash chromatography was 134 performed with the Reveleris Flash System (GRACE). 1H and 13C NMR spectra were obtained on a BRUKER 135 Advance III 400 spectrometer. All the solvents and chemicals used were of analytical grade. Optical rotation was 136 taken with a JASCO P-2000 series polarimeter. 136 taken with a JASCO P-2000 series polarimeter. 137 Insecticidal bioactivity 138 For the bioassays, artificial diet test cages were prepared as described by Sadeghi et al. [30] 100 µL of liquid 139 artificial diet was sealed between two layers of parafilm. Ten neonate aphids were placed on these layers of the 140 parafilm and to prevent the escape of aphids, the cages were covered with a hollow plastic ring having a ventilated 141 lid. These cages were placed in an inverted position in six aerated well plates. Five concentrations were used for 142 each treatment against the aphids. A stock solution of 1% was prepared by adding 1 mg of each fraction in 100 µL 143 of water. In case of the reversed-phase flash fractions, five concentrations with 50, 25, 12.5, 6.3 and 3.1 ppm and in 144 case of the prep-LC and acidic extraction fractions, five concentrations of 5, 2.5, 1.3, 0.7 and 0.3 ppm were prepared 145 by diluting the stock solution with the artificial diet of aphids. For each concentration, a final volume of 300 µL was 146 made to carry out three replications of each treatment (100 µL for each replication). Pure isolated and identified 147 active compound was analyzed in eight different concentrations including 50, 25, 12.5, 6.3, 3.1, 1.6, 0.8 and 0.4 ppm 148 by using a stock solution of 1 mg of compound in 100 µL of water. The untreated artificial diet was used as a control 149 and three replications were used for each treatment in all the bioassays. Mortality was analyzed after 24 h of each 150 treatment. 138 For the bioassays, artificial diet test cages were prepared as described by Sadeghi et al. [30] 100 µL of liquid 139 artificial diet was sealed between two layers of parafilm. Ten neonate aphids were placed on these layers of the 140 parafilm and to prevent the escape of aphids, the cages were covered with a hollow plastic ring having a ventilated 141 lid. These cages were placed in an inverted position in six aerated well plates. Five concentrations were used for 142 each treatment against the aphids. A stock solution of 1% was prepared by adding 1 mg of each fraction in 100 µL 143 of water. 137 Insecticidal bioactivity In case of the reversed-phase flash fractions, five concentrations with 50, 25, 12.5, 6.3 and 3.1 ppm and in 144 case of the prep-LC and acidic extraction fractions, five concentrations of 5, 2.5, 1.3, 0.7 and 0.3 ppm were prepared 145 by diluting the stock solution with the artificial diet of aphids. For each concentration, a final volume of 300 µL was 146 made to carry out three replications of each treatment (100 µL for each replication). Pure isolated and identified 147 active compound was analyzed in eight different concentrations including 50, 25, 12.5, 6.3, 3.1, 1.6, 0.8 and 0.4 ppm 148 by using a stock solution of 1 mg of compound in 100 µL of water. The untreated artificial diet was used as a control 149 and three replications were used for each treatment in all the bioassays. Mortality was analyzed after 24 h of each 150 treatment. 151 Additionally, the growth of the surviving aphids exposed to 0.4 ppm of the active compound for 24 h was 151 Additionally, the growth of the surviving aphids exposed to 0.4 ppm of the active compound for 24 h was 152 followed for 9 days (on the same treated diet) in comparison to the untreated aphids. 151 Additionally, the growth of the surviving aphids exposed to 0.4 ppm of the active compound for 24 h was 152 followed for 9 days (on the same treated diet) in comparison to the untreated aphids. 151 Additionally, the growth of the surviving aphids exposed to 0.4 ppm of the active compound for 24 h was 152 followed for 9 days (on the same treated diet) in comparison to the untreated aphids. 153 Data analysis 154 For statistical analysis, Probit analysis of mortality vs. concentration using POLO-Plus program version 2 155 was conducted and the lethal concentrations (LC50, LC90) and their corresponding 95% confidence intervals (95% 156 CI) were estimated for each fraction. When the 95% CI’s did not overlap, LC’s were considered to be significantly 157 different. 9 9 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 159 Bioactivity of fractions from the butanol extract of I. rugosus 160 Bioactivity of the fourteen fractions (1A-14A) obtained through the first reversed-phase flash chromatography of 161 500 mg of butanol extract of I. rugosus was analyzed for 24 h against A. pisum. Except fractions 8A, 9A, 11A, 13A 162 and 14A, all the other fractions showed considerable toxic effects against A. pisum. Fraction 3A (LC50 = 2.1 ppm and 163 LC90 = 29.5 ppm) had the highest activity as compared to all other fractions, followed by fraction 5A (LC50 = 3.3 164 ppm and LC90 = 50 ppm). Fraction, 1A (LC50 = 5.5 ppm and LC90 = 66 ppm), 2A (LC50 = 8.9 ppm and LC90 = 81 165 ppm), 4A (LC50 = 6.8 ppm and LC90 = 112 ppm) and 6A (LC50 = 17.8 ppm and LC90 = 187 ppm) gave considerable 166 mortality. Fraction 7A (LC50 = 74 ppm and LC90 = 267 ppm) showed lower mortality. Moderate toxicity was 167 observed with fraction 10A (LC50 = 36 ppm and LC90 = 53 ppm) and 12A (LC50 = 51 ppm and LC90 = 109 ppm) 168 (Table 5). 160 Bioactivity of the fourteen fractions (1A-14A) obtained through the first reversed-phase flash chromatography of 161 500 mg of butanol extract of I. rugosus was analyzed for 24 h against A. pisum. Except fractions 8A, 9A, 11A, 13A 162 and 14A, all the other fractions showed considerable toxic effects against A. pisum. Fraction 3A (LC50 = 2.1 ppm and 163 LC90 = 29.5 ppm) had the highest activity as compared to all other fractions, followed by fraction 5A (LC50 = 3.3 164 ppm and LC90 = 50 ppm). Fraction, 1A (LC50 = 5.5 ppm and LC90 = 66 ppm), 2A (LC50 = 8.9 ppm and LC90 = 81 165 ppm), 4A (LC50 = 6.8 ppm and LC90 = 112 ppm) and 6A (LC50 = 17.8 ppm and LC90 = 187 ppm) gave considerable 166 mortality. Fraction 7A (LC50 = 74 ppm and LC90 = 267 ppm) showed lower mortality. Moderate toxicity was 167 observed with fraction 10A (LC50 = 36 ppm and LC90 = 53 ppm) and 12A (LC50 = 51 ppm and LC90 = 109 ppm) 168 (Table 5). 10 169 Table 5. as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, 3A 159 Bioactivity of fractions from the butanol extract of I. rugosus ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 159 Bioactivity of fractions from the butanol extract of I. rugosus Toxicity of subfractions of the butanol fraction from first reversed-phase flash chromatography against newborn (˂ 24 h old) Acyrthosiphon 170 pisum nymphs following 24 h exposure to artificial diet containing different concentrations of subfractions 171 172 173 P 174 175 176 177 178 179 180 181 182 183 184 185 186 187 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and Fractions LC50 (95% CI) ppm Ratio LC90 (95% CI) ppm Ratio Slope ± SE Chi-Square HF 1A 5.5 (3-8) a 2.6 66 (37-211) a 2.2 1.1 ± 0.3 7.1 0.5 2A 8.9 (6.1-12) a 4.2 81 (47-231) a 2.7 1.3 ± 0.3 5.6 0.4 3A 2.1 (0.6-3.8) a 1.0 30 (18-85) a 1.0 1.1± 0.3 7.5 0.6 4A 6.8 (3.8-10) a 3.2 112.2 (54-561) a 3.8 1.1 ± 0.3 4.6 0.4 5A 3.3 (1.3-5.4) a 1.6 50 (28-176) a 1.7 1.1 ± 0.3 10.1 0.8 6A 18 (13-27) b 8.5 187 (90-808) a 6.3 1.3 ± 0.3 3.8 0.3 7A 74 (52-169) c 35.3 267 (131-1651) a 9.1 2.3 ± 0.6 8.1 0.6 8A - - - - 1.7 ± 0.7 7.0 0.5 9A - - - - 2.0 ± 1.3 4.7 0.4 10A 36 (33-40) d 17.2 52.5 (46-64) a 1.8 8.0 ± 1.4 2.8 0.2 11A - - - - 1.6 ± 0.6 8.5 0.7 12A 51 (43-71) c 24.5 109 (77-241) a 3.7 3.9 ± 1.0 2.2 0.2 13A - - - - 1.5 ± 1.2 6.6 0.5 14A - - - - 2 ± 1.3 4.7 0.4 169 Table 5. Toxicity of subfractions of the butanol fraction from first reversed-phase flash chromatography against newborn (˂ 24 h old) Acyrthosiphon 170 pisum nymphs following 24 h exposure to artificial diet containing different concentrations of subfractions oxicity of subfractions of the butanol fraction from first reversed-phase flash chromatography against newborn (˂ 24 h old) Acyrthosiphon phs following 24 h exposure to artificial diet containing different concentrations of subfractions 11 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. 189 Bioactivity of subfractions from fraction 3A collected through prep- 189 Bioactivity of subfractions from fraction 3A collected through prep- 190 LC 191 The three collected subfractions (3A-1, 3A-2 and 3A-3) of 3A were analyzed against A. pisum for 24 h. Fraction 3A- 192 1 and fraction 3A-2 gave negligible toxic effects (no LC50 and LC90). Fraction 3A-3 was the most toxic fraction 193 analyzed against A. pisum with low LC’s (LC50 = 1 ppm and LC90 = 14 ppm) (Table 6). 12 194 Table 6. Toxicity of subfractions of fraction 3A against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs following 24 h exposure to artificial diet 195 containing different concentrations of subfractions 196 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 197 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, 3A-3 Fractions LC50 (95% CI) ppm Ratio LC90 (95% CI) ppm Ratio Slope ± SE Chi-Square HF 3A-1 - - - - 2.0 ± 1.3 4.9 0.4 3A-2 - - - - 1.5 ± 1.2 6.6 0.5 3A-3 1 (0.6-1.6) a 1 14 (6.1-97) a 1 1.1± 0.3 14.8 1.1 94 Table 6. Toxicity of subfractions of fraction 3A against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs following 24 h exposure to artificial diet 95 containing different concentrations of subfractions 196 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 197 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. 189 Bioactivity of subfractions from fraction 3A collected through prep- Ratio, LCx, fraction/LCx, 3A-3 96 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 97 as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 197 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, 3A-3 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, 3A-3 13 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 198 Spectroscopic analysis of fraction 3A-3 199 Out of three subfractions of 3A (3A-1, 3A-2 and 3A-3), fraction 3A-3 was the most bioactive fraction against A. 200 pisum. This fraction 3A-3 was analyzed through 1H NMR which confirmed that the bioactive fraction 3A-3 201 contained rosmarinic acid. Different gradients were used to purify the compound but during different Prep-LC runs, 202 the chromatographic behavior, that is, peak shape and position, of this fraction was inconsistent. Therefore, the 203 reversed-phase flash chromatography was repeated with 5 g of butanol fraction of I. rugosus in order to get the most 204 bioactive compound in pure form. d 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 206 phase flash chromatography It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 206 phase flash chromatography 207 Six fractions (1B-6B) obtained through second reversed-phase flash chromatography of the butanol extract of I. 208 rugosus, were analyzed against A. pisum for 24 h. Out of the six fractions analyzed, fraction 4B, 5B and 6B showed 209 negligible toxicity (no LC50 and LC90). Fraction 1B was more toxic (LC50 = 2.5 ppm and LC90 = 28 ppm) and 210 moderate toxicity was observed for fraction 2B (LC50 = 7.5 ppm and LC90 = 71 ppm). Lower toxicity was found for 211 fraction 3B (LC50 = 16.3 ppm and LC90 = 101 ppm) (Table 7). 14 212 Table 7. Toxicity of subfractions of the butanol fraction from second reversed-phase flash chromatography against newborn (˂24 h old) Acyrthosiphon 213 pisum nymphs following 24 h exposure to artificial diet containing different concentrations of subfractions Table 7. Toxicity of subfractions of the butanol fraction from second reversed-phase flash chromatography against newborn (˂24 h old) Acyrthosiphon pisum nymphs following 24 h exposure to artificial diet containing different concentrations of subfractions 212 Table 7. Toxicity of subfractions of the butanol fraction from second reversed-phase flash chromatography against newborn (˂24 h old) Acyrthosiphon 213 pisum nymphs following 24 h exposure to artificial diet containing different concentrations of subfractions 214 215 216 217 218 219 220 221 222 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 223 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, 1B Fractions LC50 (95% CI) ppm Ratio LC90 (95% CI) ppm Ratio Slope ± SE Chi-Square HF 1B 2.5 (1-4.1) a 1.0 28 (18-69) a 1 1.2 ± 0.3 11.4 0.9 2B 7.5 (4.3-11) b 3.0 71 (38-280) a 2.5 1.3 ± 0.3 16.5 1.3 3B 16 (11-26) c 6.5 101 (52-417) a 3.6 1.6± 0.3 22.3 1.7 4B - - - - 1.0 ± 0.3 25.3 2.0 5B - - - - 1.5 ± 1.2 6.6 0.5 6B - - - - 1.8 ± 0.7 6.5 0.5 15 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 225 extraction CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 241 Fig 1. Mass spectra (negative mode electrospray ionization) of rosmarinic acid obtained via HPLC-MS with a 241 Fig 1. Mass spectra (negative mode electrospray ionization) of rosmarinic acid obtained via HPLC-MS with a 242 pseudo molecular ion at m/z value of 359 (a) Isolated rosmarinic acid (b) Commercial rosmarinic acid 242 pseudo molecular ion at m/z value of 359 (a) Isolated rosmarinic acid (b) Commercial rosmarinic acid 243 Optical rotation and 1H and 13C NMR 244 Brown crystals; +78.0° (c 0.233, MeOH); 1H NMR (400 MHz, CD3OD): δ 3.01 (1H, dd, J = 8.3, 14.3 Hz [α]24 D 245 H7a), 3.10 (1H, dd, J = 4.4, 14.3 Hz, H7b), 5.19 (1H, dd, J = 4.4, 8.3 Hz, H8), 6.27 (1H, d, J = 15.9, H17), 6.61 (1H 246 dd, J = 2.0, 8.0 Hz, H6), 6.70 (1H, d, J = 8.0 Hz, H5), 6.75 (1H, d, J = 2.0 Hz, H2), 6.78 (1H, d, J = 8.2 Hz, H14), 6.9 247 (1H, dd, J = 2.0, 8.2 Hz, H15), 7.04 (1H, d, J = 2.0 Hz, H11), 7.55 (1H, d, J = 15.9 Hz, H16);13C NMR (100 MHz 248 CD3OD): δ 37.9 (C7), 74.6 (C8), 114.4 (C17), 115.2 (C11), 116.3 (C5), 116.5 (C14), 117.6 (C2), 121.8 (C6), 123. 249 (C15), 127.7 (C10), 129.2 (C1), 145.3 (C4), 146.2 (C3), 146.8 (C12), 147.7 (C16), 149.7 (C13), 168.4 (C18), 173.5 (C9 250 ESI-MS: m/z (%) 359 (M-H+, 100). Optical rotation and NMR data were in accordance with the literature (Fig 2 251 [31, 32]. 252 Fig 2. Structure of rosmarinic acid isolated from I. rugosus 253 Bioactivity of I. rugosus rosmarinic acid and commercial rosmarinic 254 acid 255 Rosmarinic acid isolated from I. rugosus and commercial rosmarinic acid (Sigma Aldrich) were analyzed against A 256 pisum for their pesticidal activity for 24 h. Both I. rugosus rosmarinic acid (RA) (LC50 = 0.2 ppm and LC90 = 5. 257 ppm) and commercial RA (LC50 = 0.2 ppm and LC90 = 14 ppm) gave similar toxic effects (Table 9). 233 Identification of the most bioactive compound 234 Out of the two phases of acidic extraction, the ethyl acetate phase fraction was the most active. After removing ethyl 235 acetate azeotropically, this fraction was analyzed and the active compound was identified as rosmarinic acid through 236 HPLC-MS, optical rotation measurement and 1H and 13C NMR spectroscopy. 234 Out of the two phases of acidic extraction, the ethyl acetate phase fraction was the most active. After removing ethyl 235 acetate azeotropically, this fraction was analyzed and the active compound was identified as rosmarinic acid through 236 HPLC-MS, optical rotation measurement and 1H and 13C NMR spectroscopy. 252 Fig 2. Structure of rosmarinic acid isolated from I. rugosus 253 Bioactivity of I. rugosus rosmarinic acid and commercial rosmarinic 253 Bioactivity of I. rugosus rosmarinic acid and commercial rosmarinic 225 extraction 226 Both collected phases of acidic extraction were analyzed for their insecticidal potential through bioassay against A. 227 pisum for 24 h. The aqueous phase gave negligible toxic effect (no LC50 and LC90) while the ethyl acetate phase 228 showed more toxicity (LC50 = 0.2 ppm and LC90 = 9.2 ppm) (Table 8). 16 229 Table 8. Toxicity of ethyl acetate and aqueous phase of acidic extraction against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs following 24 h 230 exposure to artificial diet containing different concentrations of both phases 231 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 232 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, ethyl acetate Fractions LC50 (95% CI) ppm Ratio LC90 (95% CI) ppm Ratio Slope ± SE Chi-Square HF Aqueous - - - - 1.5 ± 1.2 6.6 0.5 Ethyl acetate 0.2 (0.04-0.5) a 1 9.2 (3.9-13)a 1 0.8 ± 0.3 4.2 0.3 229 Table 8. Toxicity of ethyl acetate and aqueous phase of acidic extraction against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs following 24 h 230 exposure to artificial diet containing different concentrations of both phases Table 8. Toxicity of ethyl acetate and aqueous phase of acidic extraction against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs following 24 h exposure to artificial diet containing different concentrations of both phases LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 231 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 232 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, fraction/LCx, ethyl acetate 17 17 . 237 HPLC-MS 238 Both isolated and commercial rosmarinic acid (Sigma Aldrich) had the same peak appearance in the HPLC-MS 239 chromatograms with the same solvent gradient. Both had a pseudo-molecular ion with an m/z value of 359 with 240 negative mode electrospray ionization which confirmed that it was rosmarinic acid (Fig 1). 238 Both isolated and commercial rosmarinic acid (Sigma Aldrich) had the same peak appearance in the HPLC-MS 239 chromatograms with the same solvent gradient. Both had a pseudo-molecular ion with an m/z value of 359 with 240 negative mode electrospray ionization which confirmed that it was rosmarinic acid (Fig 1). 241 Fig 1. Mass spectra (negative mode electrospray ionization) of rosmarinic acid obtained via HPLC-MS with a 243 Optical rotation and 1H and 13C NMR 244 Brown crystals; +78.0° (c 0.233, MeOH); 1H NMR (400 MHz, CD3OD): δ 3.01 (1H, dd, J = 8.3, 14.3 Hz, [α]24 D 245 H7a), 3.10 (1H, dd, J = 4.4, 14.3 Hz, H7b), 5.19 (1H, dd, J = 4.4, 8.3 Hz, H8), 6.27 (1H, d, J = 15.9, H17), 6.61 (1H, 246 dd, J = 2.0, 8.0 Hz, H6), 6.70 (1H, d, J = 8.0 Hz, H5), 6.75 (1H, d, J = 2.0 Hz, H2), 6.78 (1H, d, J = 8.2 Hz, H14), 6.95 247 (1H, dd, J = 2.0, 8.2 Hz, H15), 7.04 (1H, d, J = 2.0 Hz, H11), 7.55 (1H, d, J = 15.9 Hz, H16);13C NMR (100 MHz, 248 CD3OD): δ 37.9 (C7), 74.6 (C8), 114.4 (C17), 115.2 (C11), 116.3 (C5), 116.5 (C14), 117.6 (C2), 121.8 (C6), 123.2 249 (C15), 127.7 (C10), 129.2 (C1), 145.3 (C4), 146.2 (C3), 146.8 (C12), 147.7 (C16), 149.7 (C13), 168.4 (C18), 173.5 (C9); 250 ESI-MS: m/z (%) 359 (M-H+, 100). Optical rotation and NMR data were in accordance with the literature (Fig 2) 251 [31, 32]. 253 Bioactivity of I. rugosus rosmarinic acid and commercial rosmarinic 255 Rosmarinic acid isolated from I. rugosus and commercial rosmarinic acid (Sigma Aldrich) were analyzed against A. 256 pisum for their pesticidal activity for 24 h. Both I. rugosus rosmarinic acid (RA) (LC50 = 0.2 ppm and LC90 = 5.4 257 ppm) and commercial RA (LC50 = 0.2 ppm and LC90 = 14 ppm) gave similar toxic effects (Table 9). 255 Rosmarinic acid isolated from I. rugosus and commercial rosmarinic acid (Sigma Aldrich) were analyzed against A. 256 pisum for their pesticidal activity for 24 h. Both I. rugosus rosmarinic acid (RA) (LC50 = 0.2 ppm and LC90 = 5.4 257 ppm) and commercial RA (LC50 = 0.2 ppm and LC90 = 14 ppm) gave similar toxic effects (Table 9). 18 258 Table 9. Toxicity of isolated rosmarinic acid (RA) and commercial rosmarinic acid (RA) against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs 259 following 24 h exposure to artificial diet containing different concentrations of isolated rosmarinic acid and commercial rosmarinic acid 260 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 261 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, compound/LCx, Isodon rugosus RA Compound LC50 (95% CI) ppm Ratio LC90 (95% CI) ppm Ratio Slope ± SE Chi-Square HF Commercial RA 0.2 (0.05-0.5) a 1 14 (7.4-42) a 2.6 0.7 ± 0.2 15.5 0.7 I. rugosus RA 0.2 (0.04-0.4) a 1 5.4 (3.3-12) a 1 0.8 ± 0.2 10.5 0.5 258 Table 9. Toxicity of isolated rosmarinic acid (RA) and commercial rosmarinic acid (RA) against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs 259 following 24 h exposure to artificial diet containing different concentrations of isolated rosmarinic acid and commercial rosmarinic acid Table 9. 271 after 24 h of bioassay, (a) to (i) comparison observed for up to 9 days, all treated aphids died by day 9 271 after 24 h of bioassay, (a) to (i) comparison observed for up to 9 days, all treated aphids died by day 9 253 Bioactivity of I. rugosus rosmarinic acid and commercial rosmarinic CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 263 rosmarinic acid-treated and untreated diet after 24 h of bioassay 264 After incorporating the rosmarinic acid in aphid’s diet at 0.4 ppm, its effect on A. pisum that survived after 24 h 265 treatment, was analyzed every day for up to 9 days (on same treated diet). It was confirmed that rosmarinic acid had 266 a drastic effect on their growth. Firstly, most aphids exposed to treated diet were dead while the survivors did not 267 grow further to become adults and were thus not able to reproduce further. Fig 3 shows a comparison between 268 treated and untreated aphids. There was a clear difference between untreated and treated aphids after day 4, and by 269 day 9 the treated aphids were all dead, while the untreated aphids were still alive. 253 Bioactivity of I. rugosus rosmarinic acid and commercial rosmarinic Toxicity of isolated rosmarinic acid (RA) and commercial rosmarinic acid (RA) against newborn (˂ 24 h old) Acyrthosiphon pisum nymphs following 24 h exposure to artificial diet containing different concentrations of isolated rosmarinic acid and commercial rosmarinic acid Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, compound/LCx, Isodon rugosus RA Compound LC50 (95% CI) ppm Ratio LC90 (95% CI) ppm Ratio Slope ± SE Chi-Square HF Commercial RA 0.2 (0.05-0.5) a 1 14 (7.4-42) a 2.6 0.7 ± 0.2 15.5 0.7 I. rugosus RA 0.2 (0.04-0.4) a 1 5.4 (3.3-12) a 1 0.8 ± 0.2 10.5 0.5 260 Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and 261 heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO-PlusV2. Different letters in the same column indicate significant differences due to non-overlapping of 95% CI. Ratio, LCx, compound/LCx, Isodon rugosus RA LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and Data is presented as 50% (LC50) and 90% (LC90) lethal concentration values (both in ppm) together with their respective 95% confidence interval (95% CI), the slope ± SE of the toxicity vs concentration curve, and the Chi-Square and heterogeneity factor HF as accuracy of data fitting to probit analysis in POLO PlusV2 Different letters in the same column indicate significant differences due to non overlapping of 95% CI Ratio LCx compound/LCx Isodon rugosus RA 19 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . 272 Discussion The inconsistent chromatographic behavior with peak splitting observed could 291 have arisen from several causes; a contamination on guard or analytical column inlet, a blocked frit or a small void 292 at the column inlet (~wear). The problem of peak shifting (variable retention times) could have been due to small 293 changes in mobile composition, temperature fluctuations, column overloading or a combination of these problems 294 which could have led to different UV patterns for each run. Due to this problem, the reversed-phase flash 295 chromatography was repeated with a larger amount of the butanol fraction. Out of all the resulting subfractions (1B- 296 6B), 1B was selected with lower LC values against A. pisum. Fraction 1B was subjected to acidic extraction to get 297 two phases, aqueous and ethyl acetate. The ethyl acetate phase fraction was more active with lower LC values. After 298 removing ethyl acetate, the active principle was identified through different spectroscopic techniques as rosmarinic 299 acid. Similarly in another study, Chakraborty et al, [36] reported the isolation of caffeic acid and rosmarinic acid 300 from Basilicum polystachyon through acidic extraction with HCl followed by partitioning with ethyl acetate and 301 analyzed their antimicrobial activities. 302 This study reports the isolation and purification of rosmarinic acid (RA) from I. rugosus and its bioactivity 303 against A. pisum for the first time. There was no significant difference observed between the bioactivity depicted by 304 both isolated and commercial rosmarinic acid. I. rugosus rosmarinic acid gave LC values of LC50 = 0.2 ppm and 305 LC90 = 5.4 ppm. These are very low LC values depicted after 24 h of bioassay and such low LC values have not 306 been previously reported in any studies with compounds against A. pisum using the same feeding bioassay 307 methodology [30, 37-39, 28, 40, 41]. This means that a very low amount of rosmarinic acid can cause significant 308 toxic effects against A. pisum in 24 h. Very few insecticidal activities have been reported for rosmarinic acid. 309 Regnault-Roger et al, [42] investigated the insecticidal activities of polyphenolic compounds, isolated from five 310 plants belonging to Lamiaceae family against Acanthoscelides obtectus (Say) and observed that among all the 311 polyphenolic compounds, rosmarinic acid and luteolin-7-glucoside were more toxic. 272 Discussion 273 Screening candidate plants, purifying active ingredients, isolating and identifying the active plant constituents is 274 required to discover new bioactive natural products [33]. We applied this methodology to identify rosmarinic acid as 275 an active principle from the plant I. rugosus. Based on our previous study on the insecticidal activity of botanical 276 extracts from various plant species, we found that the extract from I. rugosus was the most toxic to A. pisum [27] 277 Further fractionation showed that the butanol fraction most likely contained the active principle. In this study we 278 used the bioactivity-guided strategy to isolate and identify the active compound as rosmarinic acid. This strategy is 279 interesting and has been used in previous studies to identify bioactive compounds. For example, the butanol fraction 280 from Citrullus colocynthis was reported to be active against the black legume aphid, Aphis craccivora, and through 281 the bioactivity-guided isolation strategy, the active principle, 2-O-ß-D-glucopyranosylcucurbitacin E, was 282 successfully isolated [34]. Similarly, in another study involving bioactivity-guided isolation, the active principle, 283 ailanthone, was isolated from the aqueous fraction of Ailanthus altissima against A. pisum [35]. 284 In this study, the butanol fraction was subfractionated through reversed-phase flash chromatography. After 285 bioactivity testing of all the resulting subfractions (1A-14A) against A. pisum, fraction 3A with lower LC values was 286 selected for further fractionation. Through prep-LC, fraction 3A was subfractionated and the resulting subfractions 287 (3A-1, 3A-2 and 3A-3) were analyzed for their bioactivity. Fraction 3A-3 with lower LC values was subjected to 20 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 288 spectroscopic analysis. 1H NMR spectroscopy confirmed that the isolated fraction contained rosmarinic acid. 289 However, due to the inconsistent chromatographic behavior during prep-LC, not enough compound could be 290 collected to record 13C NMR data. 320 Conclusion 321 In this study, I. rugosus was identified as an interesting source for a botanical insecticide against A. pisum. 322 Following bioactivity-guided selection, rosmarinic acid was isolated and identified through spectroscopic analysis as 323 the bioactive compound in the I. rugosus extract. Based on the bioassay results, either the extracts from I. rugosus or 324 the isolated insecticidal compound, rosmarinic acid could be exploited to develop potent aphicides, because of the 325 high mortality of aphids caused at very low rosmarinic acid concentrations. This potential botanical insecticide may 326 fit well in integrated pest management programs designed to control aphids. Considering that I. rugosus is already 327 used for medicinal purposes, it is expected to be safer compared to the current conventional pesticides used to 328 control aphids. Also, rosmarinic acid is known to reduce genotoxic effects induced by chemicals, which is contrary 329 to some currently used toxic synthetic pesticides that could induce genotoxic effects in consumers. While this study 330 highlights the potential of I. rugosus as a possible biopesticide source against a notorious insect pest such as A. 331 pisum, it also provides the basis for further exploration and development of a formulation for effective field 332 application. 272 Discussion An interesting avenue to follow 312 for future studies will be the analyses of the underlying molecular mechanisms responsible for the cause of mortality 313 in rosmarinic acid-treated aphids. 314 Additionally, a comparison between the growth of surviving aphids exposed to rosmarinic acid-treated and 315 untreated diet after 24 h of bioassay was analyzed. It was clearly observed that the growth of surviving A. pisum 21 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/591271 doi: bioRxiv preprint 316 nymphs stopped after 48 h of exposure to rosmarinic acid-treated diet, resulting in a size reduction and ultimately 317 death as compared to aphids exposed to an untreated diet. A similar observation was made by Sadeghi et al, [30] 318 who observed that the aphid size was reduced after 48 h of exposure to novel biorational insecticides, flonicamid 319 and pymetrozine, and mortality was observed after 72 h. 316 nymphs stopped after 48 h of exposure to rosmarinic acid-treated diet, resulting in a size reduction and ultimately 317 death as compared to aphids exposed to an untreated diet. A similar observation was made by Sadeghi et al, [30] 318 who observed that the aphid size was reduced after 48 h of exposure to novel biorational insecticides, flonicamid 319 and pymetrozine, and mortality was observed after 72 h. 333 Acknowledgments 334 The authors are highly thankful to the PhD scholar, Kleber Pereira at the Faculty of Bioscience Engineering, Ghent 335 University, Ghent, for maintaining and taking care of aphid colonies in the laboratory. We are grateful to Dr. Irum 336 Shehzadi and other colleagues at the Department of Environmental Sciences, COMSATS University Islamabad, 337 Abbottabad Campus, Pakistan, for their helpful support throughout this research project. 334 The authors are highly thankful to the PhD scholar, Kleber Pereira at the Faculty of Bios 337 Abbottabad Campus, Pakistan, for their helpful support throughout this research project. 338 Author Contributions 2010; 333(6-7): 539-553. 353 6) Via S, Shaw AJ. Clonal genetic variability and short term evolution in the size and shape of pea aphids 354 Evolution. 1996; 50: 163–173. 355 7) Via S. Reproductive isolation between sympatric races of pea aphids. I Gene flow restriction and habita 356 choice. Evolution. 1999; 53:1446–1457. 357 8) Peccoud J, Simon JCH, McLaughlin HJ, Moran NA. Postpleistocene radiation of the pea aphid complex 358 revealed by rapidly evolving endosymbionts. Proc Natl Acad Sci USA. 2009a; 106: 16315–16320. 359 9) Peccoud J, Ollivier A, Plantegenest M, Simon JCHA. Continuum of genetic divergence from sympatri 360 host races to species in the pea aphid complex. Proc Natl Acad Sci USA. 2009b; 106: 7495–7500. 361 10) Sparks TC, Nauen RIRAC: mode of action classiﬁcation and insecticide resistance management. Pesti 362 Biochem Physiol. 2015; 121: 122–128. 363 11) Zhang J, Goyer C, Pelletier Y. Environmental stresses induce the expression of putative glycine-rich insec 338 Author Contributions 339 SK, CT, EB, RA, NF, SM, GS and MS conceived and designed the research. SK conducted the experiments. GS and 340 SM contributed new reagents and/or analytical tools. SK, CT and EB, MA analyzed the data. SK, CT, EB, SM, GS 341 and MS wrote the manuscript. All authors read and approved the manuscript. 339 SK, CT, EB, RA, NF, SM, GS and MS conceived and designed the research. SK conducted the experiments. GS and 340 SM contributed new reagents and/or analytical tools. SK, CT and EB, MA analyzed the data. SK, CT, EB, SM, GS 339 SK, CT, EB, RA, NF, SM, GS and MS conceived and designed the research. SK conducted the experiments. GS and 340 ib d d/ l i l l d l d h d 9 SK, CT, EB, RA, NF, SM, GS and MS conceived and designed the research. SK conducted th 0 SM contributed new reagents and/or analytical tools. SK, CT and EB, MA analyzed the data 341 and MS wrote the manuscript. 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https://openalex.org/W3122453000	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0246258&type=printable	English	null	Stress reactivity near birth affects nest building timing and offspring number and survival in the European rabbit (Oryctolagus cuniculus)	PloS one	2,021	cc-by	7,758	PLOS ONE RESEARCH ARTICLE Editor: Elissa Z. Cameron, University of Tasmania, AUSTRALIA Editor: Elissa Z. Cameron, University of Tasmania, AUSTRALIA Received: March 3, 2020 Accepted: January 18, 2021 Published: January 29, 2021 Copyright: © 2021 Benedek 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 (S1 Table). Funding: The work was supported by the EFOP- 3.6.1-16-2016-00007 and GINOP-2.3.4-15-2016- 00005 projects of the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Stress reactivity near birth affects nest building timing and offspring number and survival in the European rabbit (Oryctolagus cuniculus) Ildiko´ Benedek1, Vilmos Altbcker1, Tama´s Molna´rID2* 1 Department of Wild Biology and Ethology, Institute of Environmental Science and Nature Conservation, Szent Istva´n University, Kaposva´r, Hungary, 2 Department of Nature Conservation and Environmental Management, Institute of Environmental Science and Nature Conservation, Szent Istva´n University, Kaposva´r, Hungary 1 Department of Wild Biology and Ethology, Institute of Environmental Science and Nature Conservation, Szent Istva´n University, Kaposva´r, Hungary, 2 Department of Nature Conservation and Environmental Management, Institute of Environmental Science and Nature Conservation, Szent Istva´n University, Kaposva´r, Hungary a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * molnart75@gmail.com * molnart75@gmail.com * molnart75@gmail.com OPEN ACCESS The physiological response to stressors has great importance, and its variance has an adaptive role in the survival of individuals. This study describes the effects of stress-axis activation on maternal behavior during the birthing process (parturition) in captive rabbits (Oryctolagus cuniculus). In this species, chances of survival are strongly influenced by nest quality. Thus, maternal care is initiated with nest preparation in late pregnancy, which itself is subject to strict and complex hormonal regulation. Among these hormones, progesterone is one of the most dominant in the process of nest construction. We have demonstrated that its level is altered by the level of cortisol elevation in the animal in question, potentially hav- ing an influence on the preparation of the nest for the newborn kittens. We found that does that had a constant and un-elevated level of cortisol metabolite while delivering their litters performed better than those individuals that showed an increased corticoid response around parturition. The latter group exhibited a perceptible delay in the building of their nests, and in addition, further losses were also experienced in their already smaller litters. As the quality of the nest itself proved to be was in no way inferior to those of the other group, this higher kitten-mortality rate may be attributed to impaired maternal behavior. Individual variances in cortisol levels may also result in subtle changes in hormonal regulation, potentially affecting the expression of maternal behavior. We have concluded that the higher level of cortisol detected in more-sensitive does effectively disrupts the natural hormonal regulation involved in their nest-building processes. Citation: Benedek I, Altbcker V, Molna´r T (2021) Stress reactivity near birth affects nest building timing and offspring number and survival in the European rabbit (Oryctolagus cuniculus). PLoS ONE 16(1): e0246258. https://doi.org/10.1371/ journal.pone.0246258 Citation: Benedek I, Altbcker V, Molna´r T (2021) Stress reactivity near birth affects nest building timing and offspring number and survival in the European rabbit (Oryctolagus cuniculus). PLoS ONE 16(1): e0246258. https://doi.org/10.1371/ journal.pone.0246258 1 Department of Wild Biology and Ethology, Institute of Environmental Science and Nature Conservation, Szent Istva´n University, Kaposva´r, Hungary, 2 Department of Nature Conservation and Environmental Management, Institute of Environmental Science and Nature Conservation, Szent Istva´n University, Kaposva´r, Hungary PLOS ONE PLOS ONE Introduction Basic parental care, which enables offspring to survive during the period of parental depen- dence, is of vital importance in altricial species. Among mammals, maternal care prevails [1]. While numerous maternal care strategies exist, the strategy adopted is not simply a product of Competing interests: The authors have declared that no competing interests exist. 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit the number of offspring produced [2]. Multiple behavioral and physical elements constitute parental care and thus contribute to the development and success of the offspring [1,3]. Prepa- ration of the nest cavity for offspring by the mother serves to exemplify this. Over 4500 known mammalian species construct burrows [4], and in a number of them, this process of burrow construction follows a specific and defined behavioral sequence [5,6]. The European wild rabbit is a nest builder, and lives under relatively intense pressure in its natural habitat from a predation standpoint, where it is the primary food source of more than 20 mammalian and avian predators [7,8]. Unlike many other herbivores [5,9] which spend a considerable amount of time together with their offspring, the maternal behavior of this partic- ular species is limited to the time spent building the nest and one singular daily nursing visit. Any delay in nest construction may have an influence on the reproductive success of the moth- ers. Seltmann, et al. [10] reported that the rate of litter mortality around the time of birth for those mothers who began their in burrow-digging and nest-building late (usually subordinate does) increased despite having successfully completed all the steps for nest construction well within a 24 hour period. The nest-building of the rabbit is composed of a time-controlled chain of behaviors which are regulated by both external (environmental) and internal (hormonal) factors. This complex process of nest building includes the digging of the burrow itself, the gathering of grass, and formulation of nest structure, such as lining it with the mother’s own fur [11–13]. External environmental factors, e.g., poor soil quality or lack of available grass, may limit both burrow- digging and nest-building. In the case of a sociable species such as the rabbit, the social envi- ronment may also have direct impact on the animal’s reproductive success [14,15]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 Introduction In a num- ber of cases, social stresses imposed by competition among females have negative consequences on reproduction [10]. These stress processes can be reflected though the mea- surement of glucocorticoid levels (GCM (glucocorticoid metabolites)), a well-established tool for measuring stress, and may also be evaluated non-invasively from feces through analysis of the degradation product of glucocorticoids [16]. Nest-building is a process, taking place under complex hormonal regulation, which deter- mines the onset of a similarly complex series of behaviors it involves [11–13]; the major transi- tions in this process are attributed to progesterone, estrogen, and prolactin levels. The gestation period of the wild rabbit lasts for 31 to 32 days. Burrow-digging is triggered by a high β-estradiol and progesterone levels on the 25th-26th day of gestation; the nest is subsequently prepared in this burrow, generally within 2–3 days, but sometimes only hours prior to parturi- tion [10]. Along with the high level of β-estradiol, an eventual decrease in the level of proges- terone effectively terminates the process of digging and simultaneously induces the gathering of nesting material and nest preparation. On the day prior to parturition, the level of prolactin rises nearly fivefold, causing fur shedding, thus enabling the mother to use it for lining the nest [17]. In the pregnant rabbit, serum levels of corticosterone (7.3 ng/ml) and cortisol (7.1 ng/ml) are identical until the last three days of pregnancy [18]. A decreasing progesterone level together with a parallel tenfold increase in cortisol between days 26–30 [18,19] has been described as an essential change in the third trimester of gestation, when the quantity of free glucocorticoid in the blood plasma rises due to higher-intensity glucocorticoid production [20] in the case of rabbits. Although still not yet confirmed in the case of rabbits, several other domesticated species (e.g., horses, sheep, cows, pigs, dogs) have been reported to exhibit ele- vated levels of maternal cortisol among other hormonal effects leading to parturition [21]. For gestating rabbits, the level of maternal cortisol reaches its maximum on day 30 (just before par- turition) and immediately falls afterward [19]. It may be assumed that this high value is related to the initiation of parturition. Ethical approval This research was approved by the Committee on the Ethics of Animal Experiments of the Szent Istva´n University Kaposva´r Campus (permit number: MA´B/2-2/2019). The authors declare that all experiments were performed in accordance with approved guidelines and regulations. Experimental animals Examinations were carried out on 30 females of mature European wild rabbits, 10 to 12 months of age, wherein their first parturitions were compared. One female did not produce offspring, and two gave birth to their kittens on the grid (out of the nest), and thus, their previ- ously recorded data were excluded from further surveys. The rabbits taking part in the experi- ment were derived from grandparents who were caught in the wild, the offspring of which subsequently became familiar with being kept in cages and imprinted to humans after birth during the first week of lactation [27]. Due to this process, their inherent fear of humans was reduced, which was necessary to facilitate ease of handling [28]. Introduction 2 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit In addition to the ovaries and the placenta, the adrenal glands also play a significant role in the production of progesterone [22]. As early as 1971, Fajer, et al. [23] found that in female rats exposed to stress, the amounts of progesterone secreted in their adrenal glands and ovaries to be practically identical. The secretion of progesterone, similar to glucocorticoids, is con- trolled by the adrenocorticotropic hormone (ACTH) in the adrenal gland [22], making it more sensitive to stress. This may result in unique differences in the level of progesterone released by the animal. The key factor in triggering nest-building behavior in females is the marked drop in proges- terone levels towards the end of gestation [17]. Individual differences in cortisol levels may result in observable variation in the breeding process [24]. Notable individual variation was demonstrated in regard to the quality of the nest itself, subsequently affecting the successful outcome of reproduction, both in terms of size of the litter and the survival rate of the kittens [10]. It has also been documented in both humans [25] and domesticated pigs [26] that dra- matic increases in glucocorticoid levels induced by maternal stress, lead to a decreased level of vitality in the offspring. In other rodents, adrenal progesterone release under stress is of nota- ble significance. The present study aimed to investigate whether individual differences in corti- sol release prior to parturition are a cause of significant differences in the level of progesterone during birth, and thus, the timing of the rabbit’s hay-carrying behavior. In our examination, we separated our subjects into two distinct groups based on their high (HR) and low cortisol responses (LR), occurring on the day of parturition. We hypothesized that a higher cortisol level would result in the prolongation of the progesterone levels, which would consequently cause a delay in the timing of the nest-building process, thereby affecting both litter size and mortality. Experimental facility Rabbits were mated naturally, and then placed individually into cages (floor area 60 x 60 cm, height 45cm) equipped with farrowing boxes (dimensions: 40 cm × 25 cm × 31 cm). The cages were made of welded wire mesh with hand-filled self-feeders and hayracks attached to the front. A rail-sliding tray made of galvanized steel provided a manure-receptical and was installed under each cage. The cages were placed in a room with a controlled temperature and 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit photoperiod (15.4±1.6˚C, 16 hours of daily lighting), installed in two rows and separated with opaque plates. Individuals were able to hear and smell each other, but visual contact was not allowed. Drinking water came from hand-filled drip-waters. Commercial rabbit feed, water, and hay were made available to them ad libitum. Behavioral analysis For the purpose of examining behavior in regard to grass collection, we provided dry grass over the standard amount of hay (approximately 100 g) to be used as nesting material 6 days before the expected time of parturition (from day 25).Behavior of the females examined was recorded with Sony HDR-XR cameras. Hay-carrying behavior can be detected quite easily as the doe collects the long pieces of grass in her mouth, which in protruding from both sides of the mouth, resemble a mustache. Similarly, the extraction of fur is also an easily-detectable behavior, observed when the female begins pulling out fur from its chest and thighs. Inception times for the collection of grass and fur extraction were examined by instantaneous sampling. At hourly intervals, we observed and recorded whether the females had begun carrying hay in their mouths or whether they had started to pull on their fur. Approaching the day of parturition, the farrowing boxes were checked every 2–3 hours. Once new-born kittens were discovered, a sliding door separating the farrowing chamber from the cage was accessed to allow removal of the kittens. Next, kittens were handled through the top opening, the total number of kittens was recorded and among them, the number of live kittens. Nests were categorised according to the following method: once the kittens reached 21 days of age, the nest was removed from the farrowing boxes. As the bottom of the nests had become wet from urine it was necessary to dry them out first. Once dry, the total mass of the nests was weighed, and the fur and blades of grass were arranged evenly in order to get a homogeneous mix. Ten samples were taken out of this amount (a pinch weighed appr.1-1.5 g). Every sample was separated into hay and fur and weighed individually on Sartorius scales in grams to two decimal points. This process produced the hay vs. fur ratio in the ten samples, enabling us to estimate the total hay and fur mass for the entirety of the nest (S1 Table). Hormone assessment Although the primary stress hormone in the European rabbit is corticosterone, not cortisol, the cortisol levels were measured in the present study since it significantly increases in the last three days of the pregnancy [19]. Progesterone and cortisol levels were measured from feces based on their breakdown products (GCM) [29,30]. Fecal samples were collected every 24 hours (at 20:00 EET) beginning from day 28 of gestation until the day following parturition. Contamination of the feces with urine was prevented with a mesh placed in the tray under the cage. This allowed the feces to remain on the mesh while the urine flowed into the tray. Once a sample was collected, the trays underneath the cages were replaced with new, completely clean trays. Finally, fecal data was collected on that day, 2 days prior, and the day following parturi- tion, were included in the analysis. Samples were stored at -20˚C until extraction. In addition to the collected samples, additional samples were taken to validate the cortisol measurement method. Ten mothers were exposed to social stress by placing two individuals in a transport box (30 30 40 cm) for one hour. The animals were removed from the box and immobilized by hand to collect blood samples. Subsequently, 2 ml of blood was taken from the ear vein (needle size 21G) within 2 minutes. The samples were centrifuged at 3,000g for10 min, and plasma was separated and stored at -20˚C until further measurement. Twenty-four hours after blood collection, a fecal sample was taken as described above and stored at -20˚C. 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit The protocol for fecal extraction was adapted from previously published methods [29]. After freeze-drying, the samples were ground, homogenized, and mixed thoroughly. 200 mg of dry-feces was then placed in a glass vial and 1.6 ml 80% methanol and 200 μl distilled water were added to extract the hormone metabolites. The vials were capped and vortexed for 30 minutes. Samples were then centrifuged (2450 rpm, 20 min, 4˚C) and the supernatant was poured off and stored at– 55˚C. At the time of use, samples were dried in a chamber (Binder) and reconstituted with ASB buffer at a 1:1 dilution rate. Hormone assessment Unlike the original method [29] where a an EIA with a highly specific antibody to a fecal corticosterone metabolites was used, a RIA method was used for cortisol and progesterone, which were developed for hormone determi- nation in the plasma of food animals using tritium labeled hormones (cortisol and progester- one-1,2,6,7-3H(N)) as well as highly-specific polyclonal antibodies raised against cortisol- 21-HS-BSA and 11αOH progesterone11HS:BSA in rabbits. The validation of the RIA method for the fecal metabolites was performed by determining the relationship between the measured levels in the serum and the feces samples. The binding parameters of the cortisol antibody were the following: specific activity >4 TBq/mmol, the lower limit of detection: 15 fmol, affin- ity: KA = 3.0x1010 l/mol. The reconstituted antiserum binds 45% of 10 000 cpm H3 labelled cortisol. Cross-reactivity of the antiserum can be seen in S2 Table. Intra-assay variation was determined to be < 5 CV%, for both cortisol and the progesterone. Inter-assay variation was 9.63 CV% for cortisol, and 1.68 CV% for progesterone. Sensitivity was 0.433 ng/mg for corti- sol, and 0.199 ng/mg for progesterone. For the purpose of determining comparability between cortisol standards in rabbits and the concentration of cortisol in their feces, a high-concentra- tion fecal sample was diluted in a sequential manner. The relationship between fecal cortisol and the standard concentration curve was determined through linear regression, the correla- tion coefficient was 0.998 (r) and the model for it was based on the y = 0.946x + 4.781 equation. Statistical analysis For examining differences in cortisol levels during gestation and at the time of parturition and their correlations with progesterone levels at time of parturition, and the correlation between the plasma cortisol and FCM levels, linear regression was applied. Arrangement into groups based on differences in cortisol levels during gestation and at the time of parturition was car- ried out using cluster analysis (k-means cluster). The number of clusters was two, and the number of iterations was limited to 10. The differences in progesterone between the two groups were examined using a two-sample Student’s t-test both during gestation and at time of parturition, and at the time of hay stacking, for the purpose of determining the quality of the hay and fur collected and the number of the kittens born. The time of fur-stacking did not show a normal distribution; as such a non-parametric Kruskal-Wallis test was used. Kitten mortalities by group were compared with a Chi2 test. Normality of the data collected was checked using the Shapiro-Wilk- test, while their homogeneity was confirmed with the Levene- test. Differences among the groups were accepted to be statistically significant at a level of P<0.05. Statistical analysis was carried out with the SPSS 11.5 software. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 Results 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit Cortisol and progesterone measured on the day of parturition showed a moderate correla- Fig 2. Relationship between GCM on day of parturition and the changes in GCM during last 3 days of pregnancy. GCM-Glucocorticoid Metabolites, linear regression: R = 0.843, F = 61.617, P = 0.001. https://doi.org/10.1371/journal.pone.0246258.g002 Fig 2. Relationship between GCM on day of parturition and the changes in GCM during last 3 days of pregnancy. GCM-Glucocorticoid Metabolites, linear regression: R = 0.843, F = 61.617, P = 0.001. g 2. Relationship between GCM on day of parturition and the changes in GCM during last 3 days of pregnancy. CM Gl i id M b li li i R F P Fig 2. Relationship between GCM on day of parturition and the changes in GCM during last 3 days of pregnancy. GCM-Glucocorticoid Metabolites, linear regression: R = 0.843, F = 61.617, P = 0.001. https://doi.org/10.1371/journal.pone.0246258.g002 Cortisol and progesterone measured on the day of parturition showed a moderate correla- tion across all does(R = 0.567 F = 11.871 P = 0.002) (Fig 4). Cortisol and progesterone measured on the day of parturition showed a moderate correla- tion across all does(R = 0.567 F = 11.871 P = 0.002) (Fig 4). The level of progesterone during gestation did not differ between the two groups (HR and LR). The average progesterone level showed a 28% decrease in the LR group. However, this change was lower in the HR group, resulting in 554.77±310.68 and 759.89±682.25ng/mg pro- gesterone levels on the day of parturition in the LR and HR groups, respectively. Differences between the groups were not significant (t = 1.086 df = 25 P = 0.288) (Fig 5). A significant dif- ference was found, however, in the starting time for hay collection(t = -2.238 df = 25 P = 0.034), where the mean scores of both cortisol elevation-based groups averaged at 26.14 ± 31.39 hours (HR) and 76.10 ± 55.57 hours (LR). No significant timing difference was indicated in regard to fur collection (Kruskal-Wallis test Chi2 = 1.642 P = 0.200), although the 26.14 ± 31.39 hours (HR) and 76.10 ± 55.57 hours (LR). PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 Results The plasma cortisol levels and the fecal cortisol metabolite levels showed a significant correla- tion (R = 0.8). The regression model describing the relationship between the two parameters is y = 530.593x-1.221 (P = 0.005, R2 = 0.64, S1 Fig). The cortisol level measured at the time of parturition showed no correlation with the corti- sol levels measured during gestation (R = 0.022 F = 0.012 df = 26 p = 0.914) (Fig 1). However, 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit a significant correlation between the rise in cortisol levels in the last three days of gestation an th ti l l l d th d f t iti b d (R 0 843 F 61 617 Fig 1. Relationship between GCM during pregnancy and on day of parturition GCM. GCM-Glucocorticoid Metabolites, linear regression: R = 0.022, P = 0.914. https://doi.org/10.1371/journal.pone.0246258.g001 Fig 1. Relationship between GCM during pregnancy and on day of parturition GCM. GCM-Glucocorticoid Metabolites, linear regression: R = 0.022, P = 0.914. https://doi.org/10.1371/journal.pone.0246258.g001 Relationship between GCM during pregnancy and on day of parturition GCM. GCM-Glucocorticoid Metabolites, linear ion: R = 0.022, P = 0.914. Fig 1. Relationship between GCM during pregnancy and on day of parturition GCM. GCM-Glucocorticoid Metabolites, linear regression: R = 0.022, P = 0.914. https://doi.org/10.1371/journal.pone.0246258.g001 a significant correlation between the rise in cortisol levels in the last three days of gestation and the cortisol levels measured on the day of parturition was observed (R = 0.843 F = 61.617 df = 26 P = 0.001) (Fig 2). The average cortisol level at the time of parturition was 826.01±479.29 ng/mg (Fig 3) in the animals examined. The cluster analysis which was performed on the basis the difference between cortisol levels measured three days before parturition and on the day of parturition itself, divided the animals into two groupings. In some individuals (HR group), a significant increase in cortisol levels took place (925.48±424.75 ng/mg); applying to 7 rabbits (average cor- tisol level at parturition: 1462.09±511.37ng/mg). In the LR group, consisting of 20 rabbits (average cortisol level at parturition: 603.38±174.74 ng/mg), cortisol levels demonstrated a decrease (-152.09±236.55 ng/mg). PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 Results https://doi.org/10.1371/journal.pone.0246258.g004 lower in the HR group. Additionally, perinatal mortality (birthday) was significantly higher (Chi2 = 5.092 df = 1 P = 0.024) in the HR group than in the LR group. lower in the HR group. Additionally, perinatal mortality (birthday) was significantly higher (Chi2 = 5.092 df = 1 P = 0.024) in the HR group than in the LR group. Results No significant timing difference was indicated in regard to fur collection (Kruskal-Wallis test Chi2 = 1.642 P = 0.200), although the 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit HR group tended to start collecting hay earlier (3.42±3.82 hours vs. 20.95±32.86 hours for the Fig 3. GCM levels on the day of parturition within two groups distinguished by changes in their GCM levels during the 3 last days before parturition. LR- Low-response group: 20 individuals showing either a decreasing or slightly-increasing value, HR-High-response group: 7 individuals showing a substantial increase. https://doi.org/10.1371/journal.pone.0246258.g003 Fig 3. GCM levels on the day of parturition within two groups distinguished by changes in their GCM levels during the 3 last days before parturition. LR- Low-response group: 20 individuals showing either a decreasing or slightly-increasing value, HR-High-response group: 7 individuals showing a substantial increase. https://doi.org/10.1371/journal.pone.0246258.g003 HR group tended to start collecting hay earlier (3.42±3.82 hours vs. 20.95±32.86 hours for the LR group). Amounts of hay collected did not differ significantly (t = -0.558 df = 25 p = 0.582) between the two groups (HR: 146.21±37.89g vs. LR 160.10±61.51g). The amount of built fur (HR: 10.91 ±6.15g vs. LR 14.20±13.18g) was very similar in each group (t = -0.631 df = 25 P = 0.534). A significant difference was found in cortisol elevation-based groups regarding the average num- ber of kittens born (t = -2.185 df = 25 p = 0.038), as the number of kittens born (Fig 6) was 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit lower in the HR group. Additionally, perinatal mortality (birthday) was significantly higher (Chi2 = 5.092 df = 1 P = 0.024) in the HR group than in the LR group. Fig 4. Relationship between GCM and progesterone values on day of parturition. GCM-Glucocorticoid Metabolites, linear regression: R = 0.567 F = 11.871 P = 0.002. https://doi.org/10.1371/journal.pone.0246258.g004 Fig 4. Relationship between GCM and progesterone values on day of parturition. GCM-Glucocorticoid Metabolites linear Fig 4. Relationship between GCM and progesterone values on day of parturition. GCM-Glucocorticoid Metabolites, linear regression: R 0 567 F 11 871 P 0 002 Fig 4. Relationship between GCM and progesterone values on day of parturition. GCM-Glucocorticoid Metabolites, linea regression: R = 0.567 F = 11.871 P = 0.002. Discussion We studied how behavioral patterns and hormonal changes in captive rabbits interact during the breeding period in order to gain greater understanding into why significant variation exists in their breeding success. We found that although certain social interactions were prevented through individual caging, variation occurred in both their behavior as well as in those under- lying hormonal changes characteristic to certain phases of the breeding process. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 9 / 15 PLOS ONE Cortisol response and nest building behavior in wild rabbit Fig 5. Changes in progesterone levels measured during pregnancy and time of parturition in the treatment groups. LR- Low- response group, HR-High-response group, two-sample t-test, t = 1.086 df = 25 P = 0.288. https://doi.org/10.1371/journal.pone.0246258.g005 We found that changes in the level of cortisol in the last three days of gestation was a good indicator of the sensitivity of the individual subject. However, mid-gestation cortisol levels (see Figs 1 and 2) did not allow us to predict the subject’s level of cortisol elevation at the time of h l h f d f d d d [ ] l h l Fig 5. Changes in progesterone levels measured during pregnancy and time of parturition in the treatment groups. LR- Low- response group, HR-High-response group, two-sample t-test, t = 1.086 df = 25 P = 0.288. https://doi.org/10.1371/journal.pone.0246258.g005 Fig 5. Changes in progesterone levels measured during pregnancy and time of parturition in the treatment groups. LR- Low- response group, HR-High-response group, two-sample t-test, t = 1.086 df = 25 P = 0.288. https://doi.org/10.1371/journal.pone.0246258.g005 We found that changes in the level of cortisol in the last three days of gestation was a good indicator of the sensitivity of the individual subject. However, mid-gestation cortisol levels (see Figs 1 and 2) did not allow us to predict the subject’s level of cortisol elevation at the time of parturition. This result matches findings of Edwards and Boonstra [20] revealing that gluco- corticoid levels stay low until the end of gestation. Cabezas, et al. [31] has also demonstrated that European rabbits display large individual variation in their glucocorticoid levels, as mea- sured in their serum or their GCM’s. This noteworthy difference between the rabbits indicates that individuals may react to stressful situations quite differently. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 Discussion Acute stress-related progesterone secretion can be explained through the limited activity of an enzyme (cytochrome P450-C21 enzyme), which is partially responsible for building cortisol 11 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit from progesterone. As part of the progesterone is released into the bloodstream before turning into cortisol, the concentration of circulating progesterone also increases [33]. We divided our does into two groups based on their cortisol-level change measured in the last three days of gestation. Low-response rabbits (LR group), showing no increase in GCM levels at the time of parturition, and exhibited normal maternal behavior, from collection of nesting material to the creation of the nest. High-response rabbits (HR group), characterized by an increase in their GCM levels around the time of parturition, were delayed by approxi- mately 50 hours in regard to the hoarding of nesting material. This result supports the conclu- sions of a Seltmann, et al. study [10], wherein stress caused by social instability and frequent competition between groups appeared to bring about delayed burrow-digging and nest-build- ing behavior in the subordinate females, supposedly as a result of having elevated glucocorti- coid levels. In our case, the rabbits were caged individually, and nesting materials were freely available to them, ad libitum, resulting in the minimalisation of social stress. This allowed us to observe with clarity that certain females reacted to birthing (a new and, unexpected situation) more sensitively, and consequently, responded to their higher cortisol elevation with a delayed behavioral pattern. Gonzalez-Mariscal, et al. [11] described that the beginning of hay-collect- ing and nest-building is signaled by a drop in the level of progesterone. A higher elevation in cortisol levels, however, results in a progesterone level which also stays high, and this may delay the collection of hay. Hoffmann, et al. [34] also suggested that a change in the secretion of progesterone might cause a time shift in the burrow-digging of European rabbits kept under laboratory conditions. They found a positive correlation between burrow-digging and elevated progesterone levels as well as increased intraspecific aggressive behavior in response to admin- istered progesterone. A similar phenomenon was described by Farkas, et al. [35], who success- fully identified two peaks in relation to the gathering of nest material in domesticated rabbits. Discussion Even small differences in hormonal changes which are responsible for triggering internal chain processes around the birthing process can result in differences in reproductive PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 10 / 15 PLOS ONE Cortisol response and nest building behavior in wild rabbit Fig 6. Number of kittens in the two treatment groups. LR- Low-response group, HR-High-response group, Chi2 test Chi2 = 5.092 df = 1 P = 0.024. https://doi.org/10.1371/journal.pone.0246258.g006 Fig 6. Number of kittens in the two treatment groups. LR- Low-response group, HR-High-response group, Chi2 te Chi2 = 5.092 df = 1 P = 0.024. https://doi.org/10.1371/journal.pone.0246258.g006 Fig 6. Number of kittens in the two treatment groups. LR- Low-response group, HR-High-response group, Chi2 test, Chi2 = 5.092 df = 1 P = 0.024. https://doi org/10 1371/journal pone 0246258 g006 Fig 6. Number of kittens in the two treatment groups. LR- Low-response group, HR-High-response group, Chi2 test, Chi2 = 5.092 df = 1 P = 0.024. https://doi.org/10.1371/journal.pone.0246258.g006 https://doi.org/10.1371/journal.pone.0246258.g006 performance. The change in cortisol levels (Figs 4 and 5) observed in our rabbits corresponded to the progesterone production of the pregnant animal, as levels of cortisol and progesterone change on the day of parturition. We may assume that the positive correlation between proges- terone and cortisol around the time of parturition was due to the higher cortisol elevation induced secretion of progesterone stored in the adrenal gland. This is due to the fact that not only ovaries, but also the adrenal glands, yield a significant amount of progesterone in lago- morphs, as well as in humans. Fajer, et al. [23] described, as early as 1971, that ACTH regulates the production of progesterone in the adrenal gland. In female rats exposed to mild stress, pro- gesterone secretion becomes as intensive in the adrenal gland as in the ovary. A similar result was published by Yoshida and Nakao [32] on milk cows and by Beaulieu-McCoy et al. [33] on the Californian sea lion (Zalophus californianus), where a positive correlation was found between cortisol and progesterone levels subsequent to a stress event. This may be explained by considering the metabolic pathway in question, as progesterone is a precursor of cortisol. Author Contributions Conceptualization: Ildiko´ Benedek, Tama´s Molna´r. Data curation: Ildiko´ Benedek, Tama´s Molna´r. Funding acquisition: Tama´s Molna´r. Investigation: Ildiko´ Benedek, Tama´s Molna´r. Methodology: Ildiko´ Benedek, Vilmos Altbcker, Tama´s Molna´r. Project administration: Ildiko´ Benedek. Resources: Vilmos Altbcker. Visualization: Tama´s Molna´r. Writing – original draft: Ildiko´ Benedek, Vilmos Altbcker, Tama´s Molna´r. Writing – review & editing: Ildiko´ Benedek, Tama´s Molna´r. Conceptualization: Ildiko´ Benedek, Tama´s Molna´r. Conceptualization: Ildiko´ Benedek, Tama´s Molna´r. Data curation: Ildiko´ Benedek, Tama´s Molna´r. Investigation: Ildiko´ Benedek, Tama´s Molna´r. Methodology: Ildiko´ Benedek, Vilmos Altbcker, Tama´s Molna´r. Project administration: Ildiko´ Benedek. Resources: Vilmos Altbcker. Visualization: Tama´s Molna´r. Visualization: Tama´s Molna´r. Writing – original draft: Ildiko´ Benedek, Vilmos Altbcker, Tama´s Molna´r. Writing – original draft: Ildiko´ Benedek, Vilmos Altbcker, Tama´s Molna´r. Writing – review & editing: Ildiko´ Benedek, Tama´s Molna´r. Discussion This causal explanation is congruent with our findings as well, with the exception that our animals were individually caged and therefore less exposed to social stress. Furthermore, Seltmann, S1 Fig. Relationship between plasma cortisol and fecal cortisol metabolites (FCM) col- lected from ten rabbit does. S1 Fig. Relationship between plasma cortisol and fecal cortisol metabolites (FCM) col- lected from ten rabbit does. Discussion [38] described litter size, birth body mass, and temperature as the most significant factors influencing kitten mortality. However, it was also found that nest mortality was higher in subordinate females exposed to social stress. This causal explanation is congruent with our findings as well, with the exception that our animals were individually caged and therefore less exposed to social stress. Furthermore, Seltmann, et al. [10] attributed the increased kitten mortality observed in cases of delayed burrow-digging and nest-building to be the result of stress induced by intra-sexual competition. On the basis of our present study, we offer a different causal explanation. In the ground squirrel (Urocitellus richardsonii), higher total cortisol during gestation resulted in a lower litter size [39]. It also in the HR group than in the LR litters. Ro¨del, et al. [38] described litter size, birth body mass, and temperature as the most significant factors influencing kitten mortality. However, it was also found that nest mortality was higher in subordinate females exposed to social stress. This causal explanation is congruent with our findings as well, with the exception that our animals were individually caged and therefore less exposed to social stress. Furthermore, Seltmann, et al. [10] attributed the increased kitten mortality observed in cases of delayed burrow-digging and nest-building to be the result of stress induced by intra-sexual competition. On the basis of our present study, we offer a different causal explanation. In the ground squirrel (Urocitellus richardsonii), higher total cortisol during gestation resulted in a lower litter size [39]. It also affects the sex ratio of the offspring through a positive relationship between maternal testoster- one and GCM, based on the stimulating effect of ACTH on the adrenal gland which is the pri- mary source of testosterone in females [40]. In our study, the analogous stimulatory effect of ACTH influences the timing of nest formation through progesterone release. Specifically, in addition to the exclusion of social stress, individual variance in cortisol levels point to subtle changes in hormonal regulation and may therefore affect both the timing as well as the suc- cessful outcome of maternal behavior. in the HR group than in the LR litters. Ro¨del, et al. [38] described litter size, birth body mass, and temperature as the most significant factors influencing kitten mortality. However, it was also found that nest mortality was higher in subordinate females exposed to social stress. S1 Table. The raw data of the experiment. (XLS) S1 Table. The raw data of the experiment. (XLS) S2 Table. Cross reactivity of the antiserum used for GCM measurement. (PDF) Discussion The first peak designating the carrying of nesting material, was on days 27 and 28, nearly iden- tical to the normal (LR) does in our experiment. The other peak of nest building culminated on the day of parturition, and corresponds to the results of our sensitive (HR) group. Our results highlight the fact that it is the individual sensitivity of the animal which decisively affects the behavioral response. More sensitive individuals react to emerging challenges with an increased glucocorticoid level, intense cortisol production also leading to a rise in proges- terone levels. Delays in nest-building were observable in individuals showing elevated GCM levels in response to parturition. Nonetheless, these individuals were still capable of building nests which were identical in quality and structure to the nests of does with less-elevated cortisol lev- els, who had begun their nest-building significantly earlier. Despite the short time frame involved (the final day), as well as our previous assumptions, neither the quantity of hay col- lected nor the fur extracted for the purpose of building the nest differed between the two corti- sol-response groups. In domesticated rabbits kept under laboratory conditions, Denenberg, et al. [36] found that nests made earlier were of better overall quality, when the basis of scoring was limited to the timing of hay–carrying and, the presence of grass and fur provided. In this system, nests built on the last day received low-quality scores. In our study, we demonstrated nest composition to be the same in this regard. Unlike in our experiment, under natural cir- cumstances in the wild, nest materials are not always at the animals’ disposal. We also found that does with higher GCM values around the time of parturition delivered smaller litters, and there were also further losses within these litters. In the European rabbit, 54% of pre-weaning mortality occurs within the first 12 hours, and 70% within the first week of the kittens’ life [37]. There was a significant difference between our groups (differentiated on changes in cortisol levels), as litter size was by 1.44 kittens larger in the LR group. Addition- ally, with a higher cortisol level around the time of birth, offspring mortality was 9.6% higher 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246258 January 29, 2021 PLOS ONE Cortisol response and nest building behavior in wild rabbit in the HR group than in the LR litters. Ro¨del, et al. References 1. Royle NJ, Smiseth PT, Ko¨lliker M. What is the parental care? 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https://openalex.org/W2276202809	https://europepmc.org/articles/pmc4763309?pdf=render	English	null	The Role of Noninvasive Ventilation in Patients with “Do Not Intubate” Order in the Emergency Setting	PloS one	2,016	cc-by	4,719	RESEARCH ARTICLE Background Citation: Vilaça M, Aragão I, Cardoso T, Dias C, Cabral-Campello G (2016) The Role of Noninvasive Ventilation in Patients with “Do Not Intubate” Order in the Emergency Setting. PLoS ONE 11(2): e0149649. doi:10.1371/journal.pone.0149649 Citation: Vilaça M, Aragão I, Cardoso T, Dias C, Cabral-Campello G (2016) The Role of Noninvasive Ventilation in Patients with “Do Not Intubate” Order in the Emergency Setting. PLoS ONE 11(2): e0149649. doi:10.1371/journal.pone.0149649 Editor: Francesco Staffieri, University of Bari, ITALY Received: July 22, 2015 Accepted: February 3, 2016 Published: February 22, 2016 Copyright: © 2016 Vilaça 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. Noninvasive ventilation (NIV) is being used increasingly in patients who have a “do not intu- bate” (DNI) order. However, the impact of NIV on the clinical and health-related quality of life (HRQOL) in the emergency setting is not known, nor is its effectiveness for relieving symptoms in end-of-life care. Editor: Francesco Staffieri, University of Bari, ITALY Received: July 22, 2015 Accepted: February 3, 2016 Published: February 22, 2016 Copyright: © 2016 Vilaça 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. Marta Vilaça1, Irene Aragão2☯, Teresa Cardoso2‡, Cláudia Dias3‡, Glória Cabral- Campello2☯ 1 Medicine Integrated Master (MIM), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Oporto University (UP), Porto, Portugal, 2 Intensive Care Unit (UCIP), Oporto Hospital Center, Porto, Portugal, 3 Center for Health Technology and Services Research (CINTESIS) and Information Sciences and Decision on Health Department (CIDES), Faculty of Medicine, Oporto University (UP), Porto, Portugal a1111 ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. t il @ il ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * martamvilaca@gmail.com q y ‡ These authors also contributed equally to this work. * martamvilaca@gmail.com Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Supported, in part, by grants from the Discipline of Initiation to Clinical Investigation (Disciplina de Iniciação à Investigação Clínica) of Instituto de Ciências Biomédicas Abel Salazar and in part by ASSUCIP (Associação dos Amigos da Unidade de Cuidados Intensivos Polivalente, Hospital de Santo António, Porto, Portugal). The Role of Noninvasive Ventilation in Patients with “Do Not Intubate” Order in the Emergency Setting Marta Vilaça1*, Irene Aragão2☯, Teresa Cardoso2‡, Cláudia Dias3‡, Glória Cabral- Campello2☯ Objective The aim of this prospective study was to determine the outcome and HRQOL impact of reg- ular use of NIV outcomes on patients with a DNI order who were admitted to the emergency room department (ED). METHODS: Eligible for participation were DNI-status patients who receive NIV for acute or acute-on-chronic respiratory failure when admitted to the ED of a tertiary care, university-affiliated, 600-bed hospital between January 2014 and December 2014. Patients were divided into 2 groups: (1) those whose DNI order related to a decision to withhold therapy and (2) those for whom any treatment, including NIV, was provided for symptom relief only. HRQOL was evaluated only in group 1, using the 12-item Short Form Health Survey (SF-12). Long-term outcome was evaluated 90 days after hospital discharge by means of a telephone interview. Copyright: © 2016 Vilaça 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. Conclusion The survival rate was 49% among DNI-status patients for whom NIV was used as a treat- ment in ED, and these patients did not experience a decline in HRQOL throughout the study. NIV did not provide significant relief of symptoms in more than half the patients who receive it for that purpose. Results During the study period 1727 patients were admitted to the ED, 243 were submitted to NIV and 70 (29%) were included in the study. Twenty-nine (41%) of the 70 enrollees received NIV for symptom relief only (group2). Active cancer [7% vs 35%, p = 0,004] and neuromus- cular diseases [0% vs. 17%] were more prevalent in this group. NIV was stopped in 59% of the patients in group 2 due to lake of clinical benefit. The in-hospital mortality rate was 37% Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 1 / 9 Noninvasive Ventilation and Patients with "Do Not Intubate" Order for group 1 and 86% for group 2 0,001). Among patients who were discharged from hospital, 23% of the group 1 and all patients in group 2 died within 90 days. Relative to baseline, no significant decline in HRQOL occurred in group 1 by 90 days postdischarge. for group 1 and 86% for group 2 0,001). Among patients who were discharged from hospital, 23% of the group 1 and all patients in group 2 died within 90 days. Relative to baseline, no significant decline in HRQOL occurred in group 1 by 90 days postdischarge. Introduction Noninvasive ventilation (NIV) is being used increasingly to support patients with a “do not intu- bate” (DNI) order who underwent respiratory failure and dyspnea [1–3]. A variety of factors may drive patients to the acute-care setting when nearing the end of life. Dyspnea is one of the most distressing symptoms experienced by dying patients, and it is a common reason for such patients to seek emergency care [4]. Many underlying disease states and acute illnesses cause shortness of breath at the end of life, and management tends to be symptomatic rather than diagnostic. In this context, invasive mechanical ventilation (IMV) is considered a nonbeneficial intervention that deprives patients of their dignity and ability to recognize their family members [5–8]. NIV does not have these disadvantages and may be more effective than oxygen alone for reducing dyspnea and decreasing the doses of morphine needed in patients with end-stage cancer [3]. In addition, the use of NIV for patients with DNI orders and potentially reversible causes of respiratory failure has become more common. Among such patients, it appears that those with reversible causes of dyspnea benefit from NIV used as a life-support intervention, with no decrease in health-related quality of life (HRQOL) and no increase in anxiety, depression or symptoms related to posttraumatic stress disorder after hospital discharge [6]. However, to our knowledge, all studies that included patients with DNI order were conducted in intensive care units (ICU) setting, and many of them did not differentiate the patients who received NIV with intention to treat from the patients in palliative care who received NIV for symptom relief [6,9]. Moreover, it appears that no study has evaluated the impact of NIV on HRQOL in patients with DNI order treated in non-ICU settings. The aim of the present study was (1) to evaluate the impact of NIV on outcome and HRQOL in patients with DNI order who were admitted to the emergency department (ED) and (2) to assess whether NIV is effective for relieving symptoms in patients receiving palliative care only. Methods Eligible for participation in this prospective study were all patients admitted to the ED of a ter- tiary care, 600-bed, university-affiliated hospital between January 2014 and December 2014, who underwent NIV for acute respiratory failure (ARF) or acute on chronic respiratory failure (ACRF). The study was approved by the Hospital Ethics Committee and the Administration Board of Oporto Hospital Center (reference number 2014.075(051-DEFI/065-CES). Informed con- sent was waived for who died shortly after admission and the Hospital Ethics Committee 2 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 Noninvasive Ventilation and Patients with "Do Not Intubate" Order approved the lack of informed consent for those patients. Written informed consent was obtained from all patients who were alive on the fifth to seventh days of admission, in accor- dance with the Ethics Committee (The written consent form was approved by this Committee). Collected data were anonymized. In our hospital the Intensive Care Department is responsible for the ICU, high-dependency units (HDU) and the ED (located in the Accidents and Emergency Department). Only physi- cians with training in intensive care are permitted to work in these locations. The use of NIV for acute care is initiated only in these environments, and only patients who require chronic NIV are admitted to these wards. Specific inclusion criteria were adult age, DNI order, and use of NIV for ARF or ACRF. Patients younger than 18 years of age were excluded. Patients with a DNI order were defined as those for whom healthcare professionals believed that tracheal intubation was not a therapeutic option. The study population was divided in 2 groups. Group 1, the “withholding therapy decision” group, comprised patients for whom a decision was made by the attending physician not to start or increase a life-sustaining intervention [10] but who received NIV with survival hopes and the. Group 2, the “symptom relief only” group, comprise patients for whom only palliative care (comfort measures) was thought to be appropriate by the attending physician; these patients received NIV only to alleviate their dyspnea [9,11]. All treatment decisions were made jointly by an intensive care physician and the patient’s attending physician. The study participants were subcategorized by the type of respiratory failure present at admission. Methods Those with pH <7.35 and PaCO2> 45 mm Hg, associated with signs of respiratory distress (respiratory rate> 25 breaths / min, with the use of respiratory accessory muscles) were considered to have acute-on-chronic respiratory failure [12–16]. Those with PaO2 <60 mmHg or PaO2 / FiO2 <300, and PaCO245mmHg, were classified as having acute hypox- emic respiratory failure [17–19]. HRQOL was evaluated at admission and 90 days after hospital discharge only for patients who received NIV as a treatment (group 1). The 12-intem Short Form Health Survey (SF-12) was used for this purpose. Within 5 to 7 days after admission patients were asked to completed this survey based on the 3 months preceding the admission [6,20]. Ninety days after hospital discharge, the surviving patients were interviewed by telephone and asked to complete the SF- 12 questionnaire again to assess their current HRQOL. Patients who died during the 90-day postdischarge period were not included in the evaluation of long term HRQOL. For all groups, length of stay in the ICU/HDU and hospital was documented, along with the in-hospital and 3 months mortality rates. Clinical variables and arterial blood gas levels were determined at baseline and 1 hour after initiation of NIV. Functional status was assessed using the Karnofsky performance scale, with ratings <70 representing patients who needed help with daily activities and ratings 70 denoting patients who were independent. Results Between January 2014 and December 2014, 1727 patients were admitted to the ED, 243 of whom received NIV for acute or acute-on-chronic respiratory failure. Seventy (28.8%) of those 243 patients had a DNI order and were entered into the study. (Fig 1) Of the 70 enrolls, 29 (41.4%) received NIV for symptom relief only (group 2), and 41 (58.6%) received it as treatment (group 1). The clinical characteristics of both groups are pres- ent in Table 1. There were no significant differences in demographic or clinical characteristics between the study groups, with exception of active cancer and neuromuscular disease, which were more prevalent in group 2 (Table 1). More than two-thirds of each group had lower functional status (Karnofsky rating <70). Due to lack of clinical benefits, NIV was stopped in 19.5% of patients in group 1 an in 58.6% in group 2 (p<0.001). Almost all patients included in group 2 died in the first day of admission, and none was admitted to the ICU or HDU. Twenty-three (56%) of the patients in group 1 were admitted to the ICU or HDU. The in-hospital mortality rate was 36.6% in group 1and 86.2% in group 2. Moreover, 63.4% of the patients in group 1 were alive at discharge, 77% of whom remained alive 90 days after discharge. No patient from group 2 was alive 90 days after discharge (Table 2). Among the 41 patients included group 1, 26 (63%) were alive at hospital discharge, and 20 (49%) were alive 90 days later. Seventeen (85%) of the 20 respond to the SF-12, allowing evalu- ation of HRQOL. The interviews were conducted between 90 and 162 days after discharge (median, 104 days). Most (15%) of the patients who did not participate in the follow-up interview either declined the invitation or did not answer the telephone. In group 1, no significant difference was observed between HRQOL at admission and at day-90 postdischarge. (Table 2 and Fig 2) Statistical analysis Categorical variables were expressed as absolute and relative frequencies. Continuous variables were described as mean and standard deviation (SD) if normal distribution was present; other- wise, they were expressed as median and interquartile range (IQR). To test hypotheses in groups of equal size, we used the t test for independent groups and the analysis of variance with 1 factor, when it was appropriate to assume normal distribution of variables. Testing of non-normal distribution of continuous variables was performed with non- parametric Mann-Whitney U or Kruskal-Wallis test, depending on the nature of the hypothe- sis being tested. 3 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 Noninvasive Ventilation and Patients with "Do Not Intubate" Order We assumed that a decrease of at least 20% in the SF-12 score represented clinical signifi- cance, in accordance with Azoulay et al [6]. For all hypotheses tested, the significance level was set at p = 0.05. Data analysis was performed using the SPSS1 (version 20.0, Statistical Package for Social Sciences; IBM, Armonk, NY). PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 Discussion Several important findings from our study. We observed that DNI status was defined for nearly one-third of patients admitted to the ED who received NIV. Patients with a DNI order who were alive 90 days after hospital discharge experienced no significant decline in HRQOL from baseline (admission). Long-term (90-day) follow-up demonstrated a survival rate of 49% in group 1 (NIV used as treatment). In patients under palliative care, who represent 40% of all study patients with DNI order, NIV was stopped in more than 50% due to lack of symptom improvement. The use of NIV in non-ICU settings has been increasing despite the lack of evidence of its safety and efficacy outside the ICU. [21–24]. However, Cabrini et al [25] demonstrated that, under the supervision of a medical emergency team, NIV could be applied in a variety of non- ICU settings, with a high success rate and few complications [25]. Our study was conducted in the ED of a tertiary care, university-affiliated hospital, which is supervised by the ICU team (doctor and nurse) and provides continuous clinical monitoring. A unique aspect or our study is the grouping of patients by whether NIV was performed with an intent to treat or solely for symptom relief. To our knowledge, this distinction has not been made in other studies. Azoulay et al [6], in a multicenter prospective observational study PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 4 / 9 Noninvasive Ventilation and Patients with "Do Not Intubate" Order in the ICU setting, demonstrated that DNI order was present for one-fifth of ICU patients who received NIV, which is lower than the prevalence in our study [6]. This likely can be explained by the difference in study setting (ours was in ED). Only 56% of the patients in group 1 of our study (withholding therapy decision) were admitted to the ICU or HDU; therefore, the Fig 1. Flow chart of the study. doi:10.1371/journal.pone.0149649.g001 Fig 1. Flow chart of the study. Fig 1. Flow chart of the study. doi:10.1371/journal.pone.0149649.g001 doi:10.1371/journal.pone.0149649.g001 in the ICU setting, demonstrated that DNI order was present for one-fifth of ICU patients who received NIV, which is lower than the prevalence in our study [6]. This likely can be explained by the difference in study setting (ours was in ED). doi:10.1371/journal.pone.0149649.t001 Discussion Only 56% of the patients in group 1 of our study (withholding therapy decision) were admitted to the ICU or HDU; therefore, the Table 1. Characteristics of Patients with DNI order who received NIV. Characteristics Group 1: NIV treatment (n = 41) Group 2: Symptoms relief only (n = 29) pa Age in years, median (IQR) 82.0 (75.0–87.0) 79.0 (73.0–85.0) 0.299 Male gender, n (%) 23 (56.1%) 18 (62.1%) 0.617 Type of respiratory failure, n (%) 0.291 ACRF 34 (82.9%) 21 72.4%) ARF 7 (17.1%) 8 (27.6%) Cause of respiratory failure, n (%) COPD exacerbation 22 (53.7%) 9 (31.0%) 0.060 ACPE 15 (36.6%) 6 (20.7%) 0.153 Pneumonia 11 (26.8%) 11 (37.9%) 0.324 Sepsis 0 (0.0%) 2 (6.9%) 0.168b Others 14 (34.1%) 3 (10.3%) 0.261b Co-morbidities, n (%) Congestive heart failure (NYHA) 19 (46.3%) 9 (31.0%) 0.198 COPD 21 (51.2%) 9 (31.0%) 0.093 Active cancer (all types) 3 (7.3%) 10 (34.5%) 0.004 Neuromuscular diseases 0 (0.0%) 5 (17.2%) 0.010b Karnofsky performance rating, n (%) 0.952 70 13 (31.7%) 9 (31.0%) - <70 28 (68.3%) 20 (69.0%) - ACPE, acute cardiogenic pulmonary edema; ACRF, acute-on-chronic respiratory failure; ARF, acute respiratory failure; COPD, chronic obstructive pulmonary disease; DNI, “do not intubate”; NIV, noninvasive ventilation; NYHA, New York Heart Association. a According to Chi square test unless otherwise indicated. b According to Fisher's exact test. doi:10.1371/journal.pone.0149649.t001 Table 1. Characteristics of Patients with DNI order who received NIV. ACPE, acute cardiogenic pulmonary edema; ACRF, acute-on-chronic respiratory failure; ARF, acute respiratory failure; COPD, chronic obstructive pulmonary disease; DNI, “do not intubate”; NIV, noninvasive ventilation; NYHA, New York Heart Association. a According to Chi square test unless otherwise indicated. PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 5 / 9 doi:10.1371/journal.pone.0149649.t002 doi:10.1371/journal.pone.0149649.g002 Noninvasive Ventilation and Patients with "Do Not Intubate" Order Table 2. Outcomes of the Patients with DNI order who received NIV. Outcome Group 1:NIV treatment (n = 41) Group 2:Symptom relief only (n = 29) Pa Stop NIV, n (%) 8 (19.5%) 17 (58.6%) <0.001 Days in ICU/HDU, median (IQR) 3 (0–7) 0 (0–0) <0.001 Days in hospital, median (IQR) 10 (5–17) 1 (0–5) <0.001 Died in hospital, n (%) 15 (36.6%) 25 (86.2%) <0.001 Died within 90 days of discharge, n (%) 6 (23.0%)c 4 (100%)c 0.009b SF-12 at admission, median (IQR) 30 (27–31) (n = 17) - SF- 12 on day 90 after hospital discharge, median (IQR) 27 (26–32) (n = 17) - DNI, “do not intubate”; ICU, intensive care unit; HDU, high dependency unit; IQR, interquartile range; NIV, noninvasive ventilation. a According to Chi square test unless otherwise indicated. b According to Fisher's exact test. c These percentages are based on the number of patients discharged: n = 26 for group 1; n = 4 for group 2. doi:10 1371/journal pone 0149649 t002 Table 2. Outcomes of the Patients with DNI order who received NIV. DNI, “do not intubate”; ICU, intensive care unit; HDU, high dependency unit; IQR, interquartile range; NIV, noninvasive ventilation. a According to Chi square test unless otherwise indicated. b According to Fisher's exact test. According to Fisher s exact test. c These percentages are based on the number of patients discharged: n = 26 for group 1; n = 4 for group 2. Fig 2. HRQOL at admission and at day- 90 postdischarge in patients with a DNI order. doi:10.1371/journal.pone.0149649.g002 Fig 2. HRQOL at admission and at day- 90 postdischarge in patients with a DNI order. doi:10.1371/journal.pone.0149649.g002 doi:10.1371/journal.pone.0149649.g002 PLOS ONE \| DOI:10.1371/journal.pone.0149649 February 22, 2016 6 / 9 Noninvasive Ventilation and Patients with "Do Not Intubate" Order difference in DNI-order prevalence may be attributable to selection bias. Four our DNI-status patients who received NIV as treatment (group 1: withholding therapy), the in-hospital mortal- ity rate was similar to that found by Azoulay et al [6]. Another important finding of our study is the fact that half the patients in group 1 were alive 90 days after hospital discharge, with no significant change in HRQOL since the time of admission. Thus, we agree with Azoulay et al [6] that NIV appears to be valuable treatment for ARF or ACRF in patients who do not benefit from IMV but who may benefit from other life- supporting interventions [6]. In our study, 41% the patients with a DNI order admitted to the ED received NIV for symp- tom relief only (group 2), and only 4 (14%) of these were alive at discharge. Most patients in group 2 died on the day of hospital admission, and NIV had to be stopped in more than half of group 2, which suggests that NIV may not be an effective palliative measure in such patients. This finding differs from results of a study by Nava et al [22], who found that NIV was more effective than oxygen in reducing the dyspnea, and that the dose of morphine could be decreased in patients with end-stage cancer [23]. Therefore, future research is warranted to assess the effects of NIV as comfort therapy only, including the perceptions and satisfaction of family members and the perspectives of healthcare professionals. This study has several limita- tions. Because it is a single-center study, conducted in a tertiary care hospital with extensive NIV experience, [18], the results may not be generalizable to other settings or circumstances, and may represent underestimations or overestimations of actual data obtained in different set- tings or larger populations. Our study population was small, and became even smaller when the patients were subcategorized, many of whom did not survive through 90 days post- discharge. Therefore, the results must be interpreted with caution. Only half the patients in group 1 were alive90 days after discharge, which result in a small sample for evaluating HRQOL. Acknowledgments We are grateful to Professor Margarida Lima for all the support provided throughout the suc- cess of this study. Supporting Information S1 Table. Minimum Data Set. (PDF) However, the interview response rate was high (85%) among these survivors. Conclusion In the present study, a DNI order was present for nearly one-third of ED-admitted patients who received NIV for ARF or ACRF. Nearly half the DNI-status who received NIV as a treat- ment were alive 90 days after hospital discharge, none of whom experienced a noteworthy change in HRQOL throughout the study. Among patients who received NIV only for symptom relief, more than half did not experience significant improvement of symptoms. All patients in this group died within 90 days of discharge. 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https://openalex.org/W2580494565	https://hal.science/hal-01605112/file/corrigendum_noroy_fcim_2017_2.pdf	English	null	Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets	Frontiers in cellular and infection microbiology	2,017	cc-by	599	A corrigendum on Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaﬀeensis Reveals Candidate Type IV Eﬀectors and Putative Host Cell Targets by Noroy, C., and Meyer, D. F. (2017). Front. Cell. Infect. Microbiol. 6:204. doi: 10.3389/fcimb.2016.00204 CORRECTION published: 04 April 2017 doi: 10.3389/fcimb.2017.00120 Keywords: type IV effectors, Ehrlichia chaffeensis, comparative genomics, host–pathogen interactions, genome plasticity Keywords: type IV effectors, Ehrlichia chaffeensis, comparative genomics, host–pathogen interactions, genome plasticity In the original article, there was a mistake in the legend for Table 1 as published. The citation of the Ehrlichia chaﬀeensis GSCID genomic project was missing. The correct legend appears below. The authors apologize for this error and state that this does not change the scientiﬁc conclusions of the article in any way. Edited and reviewed by: Rey Carabeo, Washington State University, USA Correspondence: Damien F. Meyer damien.meyer@cirad.fr Edited and reviewed by: Rey Carabeo, Washington State University, USA Washington State University, USA y y TABLE 1 \| Main biological and genetic characteristics of the eight Ehrlichia chaﬀeensis strains analyzed. Adapted from Rikihisa et al. (2011). Correspondence: Damien F. Meyer damien.meyer@cirad.fr 1 CIRAD, UMR ASTRE, Guadeloupe, France, 2 INRA, UMR 1309 ASTRE, Montpellier, France, 3 Université des Antilles, Guadeloupe, France 1 CIRAD, UMR ASTRE, Guadeloupe, France, 2 INRA, UMR 1309 ASTRE, Montpellier, France, 3 Université des Antilles, Guadeloupe, France REFERENCES Received: 13 March 2017 Rikihisa, Y., Hotopp, J. C. D., and Lin, M. (2011). Sequencing and Comparison of Genomes and Transcriptome Proﬁles of Human Ehrlichiosis Agents. Available online at: http://gscid.igs.umaryland.edu/doc/whitepapers/ sequencing_and_comparison_of_genomes_and_transcriptome_proﬁles_of_human_ehrlichiosis_agents.pdf (Accessed April, 2016). Rikihisa, Y., Hotopp, J. C. D., and Lin, M. (2011). Sequencing and Comparison of Genomes and Transcriptome Proﬁles of Human Ehrlichiosis Agents. Available online at: http://gscid.igs.umaryland.edu/doc/whitepapers/ sequencing_and_comparison_of_genomes_and_transcriptome_proﬁles_of_human_ehrlichiosis_agents.pdf (Accessed April, 2016). Accepted: 24 March 2017 Published: 04 April 2017 Corrigendum: Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets Christophe Noroy 1, 2, 3 and Damien F. Meyer 1, 2* Citation: Noroy C and Meyer DF (2017) Corrigendum: Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets. Front. Cell. Infect. Microbiol. 7:120. doi: 10.3389/fcimb.2017.00120 Noroy C and Meyer DF (2017) Corrigendum: Comparative Genomics Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Corrigendum: Comparative Genomics of the Zoonotic Pathogen Ehrlichia of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Copyright © 2017 Noroy and Meyer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2017 Noroy and Meyer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. April 2017 \| Volume 7 \| Article 120 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org
https://openalex.org/W3100022931	https://www.aanda.org/articles/aa/pdf/2019/03/aa35253-19.pdf	English	null	First direct detection of an exoplanet by optical interferometry; Astrometry and K-band spectroscopy of HR8799 e	arXiv (Cornell University)	2,019	cc-by	6,344	⋆The reduced spectrum is available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc. u-strasbg.fr/viz-bin/qcat?J/A+A/623/L11. ⋆⋆Based on observations collected at the European Organisation for Astronomical Research in the Southern Hemisphere, ID 60.A-9102(G). ⋆⋆⋆51 Pegasi b Fellow. Received 12 February 2019 / Accepted 28 February 2019 Received 12 February 2019 / Accepted 28 February 2019 ABSTRACT Aims. To date, infrared interferometry at best achieved contrast ratios of a few times 10−4 on bright targets. GRAVITY, with its dual-ﬁeld mode, is now capable of high contrast observations, enabling the direct observation of exoplanets. We demonstrate the technique on HR 8799, a young planetary system composed of four known giant exoplanets. Methods. We used the GRAVITY fringe tracker to lock the fringes on the central star, and integrated oﬀ-axis on the HR 8799 e planet situated at 390 mas from the star. Data reduction included post-processing to remove the ﬂux leaking from the central star and to extract the coherent ﬂux of the planet. The inferred K band spectrum of the planet has a spectral resolution of 500. We also derive the astrometric position of the planet relative to the star with a precision on the order of 100 µas. Results. The GRAVITY astrometric measurement disfavors perfectly coplanar stable orbital solutions. A small adjustment of a few degrees to the orbital inclination of HR 8799 e can resolve the tension, implying that the orbits are close to, but not strictly coplanar. The spectrum, with a signal-to-noise ratio of ≈5 per spectral channel, is compatible with a late-type L brown dwarf. Using Exo-REM synthetic spectra, we derive a temperature of 1150 ± 50 K and a surface gravity of 104.3±0.3 cm s2. This corresponds to a radius of 1.17+0.13 −0.11 RJup and a mass of 10+7 −4 MJup, which is an independent conﬁrmation of mass estimates from evolutionary models. Our results demonstrate the power of interferometry for the direct detection and spectroscopic study of exoplanets at close angular separations from their stars. Key words. stars: individual: HR 8799 – planets and satellites: atmospheres – planet-star interactions – techniques: interferometric Open Access article, published by EDP Sciences, 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. Astronomy & Astrophysics Astronomy & Astrophysics A&A 623, L11 (2019) https://doi.org/10.1051/0004-6361/201935253 c⃝S. Lacour et al. 2019 Astronomy & Astrophysics A&A 623, L11 (2019) https://doi.org/10.1051/0004-6361/201935253 c⃝S. Lacour et al. 2019 LETTER TO THE EDITOR LETTER TO THE EDITOR First direct detection of an exoplanet by optical interferometry Astrometry and K-band spectroscopy of HR 8799 e⋆,⋆⋆ GRAVITY Collaboration: S. Lacour1,2, M. Nowak1, J. Wang20,⋆⋆⋆, O. Pfuhl2, F. Eisenhauer2, R. Abuter8, A. Amorim6,14, N. Anugu7,22, M. Benisty5, J. P. Berger5, H. Beust5, N. Blind10, M. Bonnefoy5, H. Bonnet8, P. Bourget9, W. Brandner3, A. Buron2, C. Collin1, B. Charnay1, F. Chapron1, Y. Clénet1, V. Coudé du Foresto1, P. T. de Zeeuw2,12, C. Deen2, R. Dembet1, J. Dexter2, G. Duvert5, A. Eckart4,11, N. M. Förster Schreiber2, P. Fédou1, P. Garcia7,9,14, R. Garcia Lopez15,3, F. Gao2, E. Gendron1, R. Genzel2,13, S. Gillessen2, P. Gordo6,14, A. Greenbaum16, M. Habibi2, X. Haubois9, F. Haußmann2, Th. Henning3, S. Hippler3, M. Horrobin4, Z. Hubert1, A. Jimenez Rosales2, L. Jocou5, S. Kendrew17,3, P. Kervella1, J. Kolb9, A.-M. Lagrange5, V. Lapeyrère1, J.-B. Le Bouquin 5, P. Léna1, M. Lippa2, R. Lenzen3, A.-L. Maire19,3, P. Mollière12, T. Ott2, T. Paumard1, K. Perraut5, G. Perrin1, L. Pueyo18, S. Rabien2, A. Ramírez9, C. Rau2, G. Rodríguez-Coira1, G. Rousset1, J. Sanchez-Bermudez21,3, S. Scheithauer3, N. Schuhler9, O. Straub1,2, C. Straubmeier4, E. Sturm2, L. J. Tacconi2, F. Vincent1, E. F. van Dishoeck2,12, S. von Fellenberg2, I. Wank4, I. Waisberg2, F. Widmann2, E. Wieprecht2, M. Wiest4, E. Wiezorrek2, J. Woillez8, S. Yazici2,4, D. Ziegler1, and G. Zins9 1. Introduction atmospheres. More precisely, the L-T transition is an important observable for understanding the evolution of atmospheres as a function of temperature. At lower temperatures (<1200 K), opac- ity changes due to the transition of CO to methane in chemi- cal equilibrium, and the likely disappearance of silicate and iron clouds under the photosphere, makes the spectral appearance of T-type BDs bluer. On the contrary, young giant exoplanets of temperature ≈1000 K still have redder near-infrared colors typ- ical of late-L BDs. This is explained by the relatively low sur- face gravity (g ≤104 cm s2), and hence larger scale heights, in planetary atmospheres. However, the exact cause (cloud properties and/or vertical chemical mixing) is not properly understood (Allard et al. 2012). Once the problem of cloud for- mation and chemical processes are solved (Helling et al. 2014; Moses et al. 2016), the determination of the molecular com- position of exoplanet atmospheres will become a crucial tool toward understanding the formation process of planets: the Obtaining accurate orbits, masses, and atmospheric spectra of directly imaged planets is key to determining their natures and, ultimately, their formation histories. Here we demonstrate the power of a new technique, using optical interferometry, to obtain this information for an exoplanet as close as 390 mas to its parent star. Because they are better known, the spectra of brown dwarfs (BD) are often used as references to classify the young exoplanet L11, page 1 of 6 A&A 623, L11 (2019) 0 10 20 30 40 50 60 70 Spatial frequency (M ) 10 5 10 4 10 3 10 2 10 1 100 Visibility mag=10.7 Stellar visibility incoherent flux UT1-UT2 UT1-UT3 UT2-UT3 UT1-UT4 UT2-UT4 UT3-UT4 -50 -25 0 25 50 U (M ) -50 -25 0 25 50 V (M ) HR8799e Fig. 1. Colored lines are HR 8799 e visibilities \|Vplanet\|, as deﬁned by Eq. (1), obtained from the ratio between the coherent ﬂux observed on the planet and on the star. The underlying dotted colored lines correspond to the errors estimated by the pipeline. The theoretical stellar visibility corresponds to the black dashed line (uniform disk of diameter 0.342 mas). The upper horizontal dotted black line corresponds to the observed incoherent ﬂux (stellar ﬂux leaking in the planet’s ﬁeld). The lower horizontal dotted black line corresponds to the theoretical visibility of a planet 10.7 mag fainter than the star. 1. Introduction Right inset: coverage of the spatial frequency plane, east is to the right. The arrow indicates the direction of the planet situated to the northwest of the star. Fig. 1. Colored lines are HR 8799 e visibilities \|Vplanet\|, as deﬁned by Eq. (1), obtained from the ratio between the coherent ﬂux observed on the planet and on the star. The underlying dotted colored lines correspond to the errors estimated by the pipeline. The theoretical stellar visibility corresponds to the black dashed line (uniform disk of diameter 0.342 mas). The upper horizontal dotted black line corresponds to the observed incoherent ﬂux (stellar ﬂux leaking in the planet’s ﬁeld). The lower horizontal dotted black line corresponds to the theoretical visibility of a planet 10.7 mag fainter than the star. Right inset: coverage of the spatial frequency plane, east is to the right. The arrow indicates the direction of the planet situated to the northwest of the star. Sect. 2 we present the GRAVITY observation of HR 8799 e and the data reduction. HR 8799 is a bright (K = 5.24 mag), nearby (d = 39.4 ± 0.1 pc) A5 star. We know that at least four planets are orbiting the star (the ﬁrst three discovered by Marois et al. 2008). In Sect. 3 we present a new astrometric mea- surement of the fourth planet (K = 15.9 mag), discovered in 2010 by Marois et al. (2010) at 368 ± 9 mas from its host star. The youth of the star (≈30 Myr, member of the Columba Associ- ation Malo et al. 2013), implies that the planet is still warm from its initial gravitational energy. In Sect. 4 we use the K-band spec- tra and photometry to constrain its spectral type, temperature, and radius. Finally, in Sect. 5, we summarize our results and brieﬂy address the prospects of the interferometric technique. atomic ratio or even isotope ratio will change depending on the conditions of formation. For example, the C-to-O ratio in the gas of a protoplanetary disk is predicted to increase outwards past the H2O (≈140 K), CO2 (≈50 K), or even CO (≈20 K) ice-lines (Öberg et al. 2011): C/O in the planet atmo- sphere should therefore change depending on where in a disk planets form and how much gas and how many planetesi- mals they accrete (Mordasini et al. 2016). 1. Introduction Similarly, the D-to- H ratio in planets can be linked to the accretion of icy bodies (Feuchtgruber et al. 2013) and can be seen in molecular absorp- tion spectra (Mollière & Snellen 2019). The study of cloud properties and composition requires spec- tral information, which can currently only be obtained by two means: transit spectroscopy or thermal emission spectroscopy. Transit spectroscopy is best for characterizing planets in close orbit around the host star, with puﬀy (inﬂated) irradiated atmo- spheres (Crossﬁeld 2015, and references herein). Thermal emis- sion spectroscopy is more adapted to young, self-luminous planets in orbits with a semimajor axis of a few tens of AU around the host star. Young planets are warm as they still pos- sess excess entropy tracing back to the formation process (e.g., Marleau & Cumming 2014). The diﬃculty with emission spec- troscopy is that a planet’s signal is contaminated by stellar pho- tons, which vary in time with changing observing conditions, resulting in a spatially and spectrally varying speckle pattern. One of the solutions for emission spectroscopy is to go to space to beneﬁt from a stable point spread function. Another is to use high contrast and high angular resolution observations on 8 m to 10 m class telescopes from the ground, and to deconvolve the image to remove the speckles by using spectro-spatial cor- relations. This is typically done using integral ﬁeld spectroscopy and techniques like spectral diﬀerential imaging (Rameau et al. 2015) or angular diﬀerential imaging (Marois et al. 2006). L11, page 2 of 6 2. Observations and data reduction The observations were obtained with the VLTI using the four 8 m Unit Telescopes and the GRAVITY instrument (Eisenhauer et al. 2011; Gravity Collaboration 2017) on 28 August 2018. GRAVITY can observe two objects located in the VLTI ﬁeld of view by simultaneously injecting, at each telescope coudé focus, the light of each object into a separate single-mode ﬁber. The two ﬁbers have an eﬀec- tive ﬁeld of view equal to ≈60 mas, the K-band diﬀraction limit of a single telescope (Pfuhl et al. 2014). Each object is thus interferometrically observed separately, but an ultrapre- cise laser telemetry constantly monitors the diﬀerential opti- cal path between the two objects. The ﬁrst ﬁber of GRAVITY was placed on the star for fringe tracking (Lacour et al. 2019) and phase referencing. The second ﬁber was centered sequen- tially on the planet and on the star, situated at ≈390 mas from each other. This second ﬁber fed the science spectrometer con- ﬁgured at medium resolution (R = 500). Observations of the star were used to calibrate the observation of the planet. Because of the faintness of the planet, a detector integration time (DIT) of 100 s was required. As the star is more than 10 mag brighter than the planet in K band, a DIT of 1 s was suﬃcient for the With the technique presented in this paper, we go one step further by using the resolving power of the ≈100 m baselines oﬀered by optical interferometry to distinguish between the coherent ﬂux originating from the star and from the planet. In L11, page 2 of 6 GRAVITY Collaboration: Astrometry and K-band spectroscopy of HR 8799 e and K-band spectroscopy of HR 8799 e Table 2. Astrometry on HR 8799 e. MJD ∆RA ∆Dec Covariance (mas) (mas) (mas2) 58358.190 −357.63 163.59 58358.205 −357.68 163.63 58358.217 −357.54 163.05 58358.241 −357.58 163.28 58358.255 −357.61 163.12 58358.269 −357.62 163.22 58358.282 −357.80 163.41 Global −357.64 ± 0.07 163.34 ± 0.18 −0.00668 800 600 400 200 0 200 400 R.A. Offset (mas) 400 200 0 200 400 Decl. Offset (mas) 400 300 200 100 0 100 358 357 163.0 163.5 Fig. 2. Keplerian orbit ﬁt of HR 8799 e. The black point is the GRAVITY measurement, and the gray points are from previous astrom- etry (Konopacky et al. 2016; Wang et al. 2018a). The yellow lines are 250 random draws from the posterior. 2. Observations and data reduction The orbit determination is cur- rently limited by the mas-level astrometry of the previous epochs (top inset). Image magniﬁed by ∼200x to display the uncertainties in the GRAVITY astrometry. The plotted error bars are rotated to be aligned with the principal axes of the error ellipse. Bottom inset: image magni- ﬁed by ∼2x to display the uncertainties in the previous measurements. Table 1. Observing log (data taken on 2018-Aug-28). Target UT Time DIT NDIT Seeing τ0 Airmass par. angle Planet 04:33:59 100 s 10 0.8′′ 4.9 ms 1.48 −164.3◦ Star 04:51:27 1 s 50 0.5′′ 7.1 ms 1.45 −169.6◦ Sky 04:52:56 1 s 50 0.5′′ 6.7 ms 1.45 −170.0◦ Planet 04:54:35 100 s 10 0.6′′ 6.2 ms 1.45 −170.6◦ Planet 05:11:55 100 s 10 0.6′′ 6.2 ms 1.44 −176.0◦ Sky 05:29:27 100 s 10 0.4′′ 8.5 ms 1.44 178.5◦ Planet 05:46:56 100 s 10 0.5′′ 6.3 ms 1.44 172.9◦ Planet 06:07:13 100 s 10 0.6′′ 5.6 ms 1.47 166.7◦ Star 06:24:42 1 s 50 0.8′′ 4.2 ms 1.50 161.5◦ Sky 06:26:10 1 s 50 0.8′′ 3.9 ms 1.50 161.1◦ Planet 06:28:04 100 s 10 0.7′′ 4.9 ms 1.50 160.5◦ Planet 06:45:25 100 s 10 0.7′′ 6.2 ms 1.55 155.7◦ Sky 07:04:02 100 s 10 1.0′′ 3.6 ms 1.62 150.9◦ Notes. τ0 is the coherence time in the visual (500 nm); par. angle is the parallactic angle. Table 2. Astrometry on HR 8799 e. Table 1. Observing log (data taken on 2018-Aug-28). 800 600 400 200 0 200 400 R.A. Offset (mas) 400 200 0 200 400 Decl. Offset (mas) 400 300 200 100 0 100 358 357 163.0 163.5 Notes. τ0 is the coherence time in the visual (500 nm); par. angle is the parallactic angle. science channel observations of the star. Seeing conditions were average to good. The log of the observations is presented in Table 1, where exposures on the planet are shown along the sky and stellar calibration exposures. p The frequency plane and the amplitude of the planet’s vis- ibilities are presented in Fig. 1. The colored dotted lines below the visibilities are the errors estimated by the pipeline. The mean S/N per spectral channel is ≈5. The detailed data reduction pro- cedure will be presented by Nowak et al. (in prep.). The main steps are as follows: 400 200 0 R.A. Offset (mas) Fig. 2. Keplerian orbit ﬁt of HR 8799 e. 1 The pipeline in its version 1.1.2 is currently available at https:// www.eso.org/sci/software/pipelines/gravity. 2. Observations and data reduction The black point is the GRAVITY measurement, and the gray points are from previous astrom- etry (Konopacky et al. 2016; Wang et al. 2018a). The yellow lines are 250 random draws from the posterior. The orbit determination is cur- rently limited by the mas-level astrometry of the previous epochs (top inset). Image magniﬁed by ∼200x to display the uncertainties in the GRAVITY astrometry. The plotted error bars are rotated to be aligned with the principal axes of the error ellipse. Bottom inset: image magni- ﬁed by ∼2x to display the uncertainties in the previous measurements. p 1. Extraction of the coherent ﬂux (the VISDATA) for individual ﬁles using the ESO GRAVITY pipeline1; 2. Derivation of the position of the planet with respect to the star by ﬁtting the coherent ﬂux with models of the coherent ﬂux from the star and from the planet; 3. Removal of the coherent ﬂux of stellar origin by linear decomposition on the models of step 2. This step assumes the position of the planet from step 2; 4. Normalization in phase and amplitude of the remaining coherent ﬂux by the coherent ﬂux observed on the star mul- tiplied by the theoretical visibility function of the star. This gives the complex visibility of the planet 4. Normalization in phase and amplitude of the remaining coherent ﬂux by the coherent ﬂux observed on the star mul- tiplied by the theoretical visibility function of the star. This gives the complex visibility of the planet The upper dotted black line corresponds to the average resid- ual ﬂux from the star entering the science spectrometer. The lower dotted black line corresponds to the theoretical ﬂux of an unresolved source with a 10.7 diﬀerence in magnitude. Vplanet = VISDATAplanet VISDATA∗ star × 2J1(πθstaru) πθstaru , (1) (1) 3. Relative astrometry where J1 is a Bessel function of the ﬁrst kind, of order 1; θstar is the stellar diameter; and u is the spatial frequency in rad−1. The amplitude of Vplanet is plotted as solid curves in Fig. 1; where J1 is a Bessel function of the ﬁrst kind, of order 1; θstar is the stellar diameter; and u is the spatial frequency in rad−1. The amplitude of Vplanet is plotted as solid curves in Fig. 1; 5. Retrieval of the spectrum of the planet by assuming a diam- eter for the planet and a synthetic stellar spectrum: where J1 is a Bessel function of the ﬁrst kind, of order 1; θstar is the stellar diameter; and u is the spatial frequency in rad−1. The amplitude of Vplanet is plotted as solid curves in Fig. 1; where J1 is a Bessel function of the ﬁrst kind, of order 1; θstar is the stellar diameter; and u is the spatial frequency in rad−1. The amplitude of Vplanet is plotted as solid curves in Fig. 1; 5. Retrieval of the spectrum of the planet by assuming a diam- eter for the planet and a synthetic stellar spectrum: In the same way as we can disentangle the complex coherent energy from the star and the planet, it is also possible to ﬁt the wavelength dependence of the phase, which is tantamount to measuring a separation. Each ﬁt, for each baseline, gives a χ2 minimum for an optimal optical path diﬀerence (OPD). This OPD corresponds to an angular separation projected in the direc- tion of the baseline vector. Several of these optimal OPDs are necessary to derive a position. By combining all the baselines together, we can use each exposure ﬁle separately, giving the seven optimal positions listed in Table 2. The global minimum is at ∆RA = −357.64 ± 0.07 mas and ∆Dec = 163.34 ± 0.18 mas with highly elliptical uncertainty (covariance of −0.00668 mas2). Along the longest baseline (position angle, PA = 78◦) the 1σ uncertainty is 55 µas. Orthogonal to that baseline (PA = 168◦) the uncertainty is 190 µas. The plate scale and true north error is negligible at that level (>50 µas) as the spatial frequencies are deﬁned by the physical position of the telescopes. Atmospheric dispersion is also negligible. A detailed description of the error is the stellar diameter; and u is the spatial frequency in rad . 3. Relative astrometry The red curve is the X-SHOOTER spectrum of the bro d f L h 16 A f L di t l (2015) th d t th GRAVITY l ti Th d d 2 i 2 4 ( 236◦ f f d ) Th Fig. 3. GRAVITY K band spectrum of HR8799 e at spectral resolution 500 (gray points). The red curve is the X-SHOOTER spectrum of the brown dwarf Luhman 16 A from Lodieu et al. (2015), smoothed to the GRAVITY resolution. The reduced χ2 red is 2.4 (over 236◦of freedom). The orange curve is the best ﬁt of the Exo-REM models from Charnay et al. (2018). The reduced χ2 red is 2.7. The dashed curve is the K-band GPI spectrum from Greenbaum et al. (2018). The square dots are the SPHERE photometry from Zurlo et al. (2016). 400 600 800 1000 1200 1400 1600 1800 2000 Temperature (K) 3.0 3.5 4.0 4.5 5.0 5.5 6.0 log(g) R = 5Rjup R = 2Rjup R = 1Rjup R = 0.7Rjup 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 2 red 400 600 800 1000 1200 1400 1600 1800 2000 Temperature (K) 3.0 3.5 4.0 4.5 5.0 5.5 6.0 log(g) R = 5Rjup R = 2Rjup R = 1Rjup R = 0.7Rjup 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 2 red Fig. 4. Reduced χ2 as a function of eﬀective temperature and surface gravity from a grid of Exo-REM models (Charnay et al. 2018). The yel- low contours correspond to the 5σ error, indicating a valley of possible temperatures between 1100 and 1200 K. The vertical lines correspond to the planet’s radius from the model K-band luminosity assuming a distance of 39.4 pc. terms of interferometric astrometry is presented in Lacour et al. (2014). As this astrometry is an order of magnitude more precise than the best measurements made by direct imaging instru- ments (Wang et al. 2018a), we investigate the orbital constraints provided by this datapoint. We ﬁt a single Keplerian orbit by combining this measurement with the astrometry reported in Konopacky et al. (2016) and Wang et al. (2018a). Given the assymetry in the GRAVITY measurement, we ﬁt for the location of the planet at the GRAVITY epoch in a rotated frame that is aligned with the two principal axes of the error ellipse (top inset of Fig. 2). 3. Relative astrometry We use the parallel-tempered Markov chain Monte Carlo sampler (Foreman-Mackey et al. 2013; Vousden et al. 2016) in the orbit ﬁtting code orbitize (Blunt et al. 2019) to estimate the orbital parameters and ﬁnd a semimajor axis of 16.4+2.1 −1.1 AU, an eccentricity of 0.15 ± 0.08, and an inclination of 25◦± 8◦. A single 100 µas precision point is able to signiﬁcantly constrain the position of the planet at the epoch of observation, but the determination of the planet’s veloc- ity, acceleration, and orbital properties are still dominated by the mas-level uncertainties in the previous astrometry. We therefore defer a thorough dynamical study to a time when multi-epoch orbital monitoring of the planet with VLTI/GRAVITY has been obtained. 000 00 0 Temperature (K) Fig. 4. Reduced χ2 as a function of eﬀective temperature and surface gravity from a grid of Exo-REM models (Charnay et al. 2018). The yel- low contours correspond to the 5σ error, indicating a valley of possible temperatures between 1100 and 1200 K. The vertical lines correspond to the planet’s radius from the model K-band luminosity assuming a distance of 39.4 pc. We can also compare the location of the planet measured by GRAVITY with the ∆RA = −352.6+3.1 −2.6 mas and ∆Dec = −157.9 ± 1.8 mas predicted by the dynamically stable coplanar solutions from Wang et al. (2018a). The positions are inconsis- tent by 5 mas in both axes and none of the 9792 stable copla- nar orbits are consistent with our measurement at the 3σ level. With this single astrometric point, we are therefore able to disfa- vor dynamically stable conﬁgurations in which the four planets are perfectly coplanar. Changing the inclination of HR 8799 e by ≈−2◦accounts for this 5 mas diﬀerence, and Wang et al. (2018a) did ﬁnd 14 stable non-coplanar orbits with mutual inclinations of less 8◦out of 20 million trials. We note that given the uncer- tainties in the orbital planes of the other three planets, we can- not pinpoint the mutual inclinations of the planets in this simple analysis. Continued monitoring of the orbit with GRAVITY can further constrain the planet’s orbital elements, allowing a search for dynamically stable non-coplanar orbital solutions to be com- putationally tractable and providing more accurate constraints on the masses of the multiple planets. 3. Relative astrometry The amplitude of Vplanet is plotted as solid curves in Fig. 1; p 5. Retrieval of the spectrum of the planet by assuming a diam- eter for the planet and a synthetic stellar spectrum: Fplanet = \|Vplanet\|Fstar 2J1(πθplanetu)/πθplanetu· (2) (2) The stellar diameter is assumed to be θstar = 0.342 ± 0.008 mas (Baines et al. 2012) and is plotted as the black dashed line in Fig. 1. The planet diameter is assumed to be negligible at the resolution of the interferometer. For the star, we used a BT- NextGen model (T = 7400 K, [Fe/H] = −0.5, and log(g) = 4.0) from Hauschildt et al. (1999), scaled for a K-band ﬂux of 3.191 × 10−12 Wm−2 µm−1. The stellar diameter is assumed to be θstar = 0.342 ± 0.008 mas (Baines et al. 2012) and is plotted as the black dashed line in Fig. 1. The planet diameter is assumed to be negligible at the resolution of the interferometer. For the star, we used a BT- NextGen model (T = 7400 K, [Fe/H] = −0.5, and log(g) = 4.0) from Hauschildt et al. (1999), scaled for a K-band ﬂux of 3.191 × 10−12 Wm−2 µm−1. L11, page 3 of 6 L11, page 3 of 6 A&A 623, L11 (2019) 1.9 2.0 2.1 2.2 2.3 2.4 2.5 Wavelength ( m) 0.00 0.01 0.02 0.03 F (10 14Wm 2 m 1) Luhman 16A Exo-REM T=1150K / log(g)=4.3 GPI data SPHERE photometry GRAVITY data Fig. 3. GRAVITY K band spectrum of HR8799 e at spectral resolution 500 (gray points). The red curve is the X-SHOOTER spectrum of the brown dwarf Luhman 16 A from Lodieu et al. (2015), smoothed to the GRAVITY resolution. The reduced χ2 red is 2.4 (over 236◦of freedom). The orange curve is the best ﬁt of the Exo-REM models from Charnay et al. (2018). The reduced χ2 red is 2.7. The dashed curve is the K-band GPI spectrum from Greenbaum et al. (2018). The square dots are the SPHERE photometry from Zurlo et al. (2016). 1.9 2.0 2.1 2.2 2.3 2.4 2.5 Wavelength ( m) 0.00 0.01 0.02 0.03 F (10 14Wm 2 m 1) Luhman 16A Exo-REM T=1150K / log(g)=4.3 GPI data SPHERE photometry GRAVITY data Fig. 3. GRAVITY K band spectrum of HR8799 e at spectral resolution 500 (gray points). L11, page 4 of 6 Acknowledgements. GRAVITY was developed via a collaboration of the Max Planck Institute for Extraterrestrial Physics, LESIA of Paris Observatory and IPAG of Université Grenoble Alpes/CNRS, the Max Planck Institute for Astron- omy, the University of Cologne, the Centro Multidisciplinar de Astroﬁsica 306 Gagné, J., Faherty, J. K., Cruz, K. L., et al. 2015, ApJS, 219, 33 Gravity Collaboration (Abuter, R., et al.) 2017, A&A, 602, A94 y ( , , ) , , , 9 Greenbaum, A. Z., Pueyo, L., Ruﬃo, J.-B., et al. 2018, AJ, 155, 226 Greenbaum, A. Z., Pueyo, L., Ruﬃo, J.-B., et al. 2018, AJ, 155, 2 Hauschildt, P. H., Allard, F., Ferguson, J., Baron, E., & Alexander, D. R. 1999, ApJ, 525, 871 4. Atmosphere of HR 8799 e Part of this work was supported by the European Union under ERC grant 639248 LITHIUM. J.W. is supported by the Heising-Simons Foundation 51 Pegasi b postdoc- toral fellowship. A.A., P.G., and N.A. acknowledge funding from Fundação para a Ciência e Tecnologia through grants PTDC/CTE-AST/116561/2010, SFRH/BD/52066/2012, COMPETE FCOMP-01-0124-FEDER-019965, UID/ FIS/00099/2013, and SFRH/BSAB/142940/2018. R.G.L. acknowledges funding by H2020 Marie Curie fellowship 706320. 1 LESIA, Observatoire de Paris, Université PSL, CNRS, Sorbonne Université, Univ. Paris Diderot, Sorbonne Paris Cité, 5 place Jules Janssen, 92195 Meudon, France e-mail: sylvestre.lacour@obspm.fr, mathias.nowak@obs pm.fr 5. Summary and conclusions p Helling, C., Woitke, P., Rimmer, P. B., et al. 2014, Life, 4, 142 Helling, C., Woitke, P., Rimmer, P. B., et al. 2014, Life, 4, 142 K k Q M B T S M i h B A & M i C 2013 S i Konopacky, Q. M., Barman, T. S., Macintosh, B. A., & Marois, C. 2013, Science, 339, 1398 Interferometric astrometry, an order of magnitude more accu- rate than direct imaging, opens new possibilities to study the dynamics of planetary systems. With just a single data point from GRAVITY, we can strongly disfavor perfectly coplanar stable orbits for the HR 8799 planets. As the dynamics probe the masses, formation history, and the future system architec- ture, interferometric orbital monitoring at the 10–100 µas level can signiﬁcantly improve our understanding of directly imaged systems. Konopacky, Q. M., Marois, C., Macintosh, B. A., et al. 2016, AJ, 152, 28 Lacour, S., Eisenhauer, F., Gillessen, S., et al. 2014, A&A, 567, A75 Lacour, S., Dembet, R., Abuter, R., et al. 2019, A&A, accepted [arXiv:1901.03202] Lodieu, N., Osorio, M. R. Z., Rebolo, R., et al. 2015, A&A, 581, A73 Luhman, K. L. 2013, ApJ, 767, L1 Malo, L., Doyon, R., Lafrenière, D., et al. 2013, ApJ, 762, 88 Marleau, G.-D., & Cumming, A. 2014, MNRAS, 437, 1378 Marois, C., Lafreniere, D., Doyon, R., Macintosh, B., & Nadeau, D. 2006, ApJ, 641, 556 y Based on the K-band spectrum, we conﬁrm a spectral type (≈L7), equivalent to a higher temperature BD. The discrepancy between spectral type (T > 1400 K; Schweitzer et al. 2002) and eﬀective temperature derived from wide-band photometry (T < 1200 K) can be solved by using models taking the lower surface gravity into account. It is interesting to note that the GRAVITY K-band spectrum does constrain this low surface gravity, as shown by the residual map in Fig. 4. We determine a surface gravity compatible with a 10 MJup planet. Marois, C., Macintosh, B., Barman, T., et al. 2008, Science, 322, 1348 Marois, C., Zuckerman, B., Konopacky, Q. M., Macintosh, B., & Barman, T. 2010, Nature, 468, 1080 Mollière, P., & Snellen, I. A. G. 2019, A&A, 622, A139 Mordasini, C., van Boekel, R., Mollière, P., Henning, T., & Benneke, B. 2016, ApJ, 832, 41 Moses, J. I., Marley, M. S., Zahnle, K., et al. 2016, ApJ, 829, 66 Öberg, K. I., Murray-Clay, R., & Bergin, E. A. 4. Atmosphere of HR 8799 e The GRAVITY spectrum of HR 8799 e was obtained by multi- plying the visibility of the planet with the theoretical spectrum of the star. This is following Eq. (2) and assuming θplanet = 0. The resulting spectrum is represented as the gray points in Fig. 3. The CO-band head at 2.29 µm is the most prominent feature. As already mentioned by Konopacky et al. (2013) and Wang et al. (2018b) for HR8799 c, no clear CH4 absorption is seen, in agree- ment with a typical L-type BD spectrum. Using H and K band GPI spectra, Greenbaum et al. (2018) obtained a best ﬁt with the spectrum of the brown dwarf WISE J1049-5319A (also called Luhman 16 A from Luhman 2013) of spectral type L7.5. The ﬁt is equally good with the GRAVITY spectrum, and gives a reduced χ2 red of 2.4. ed We ﬁtted the catalog of BD spectra from the Montreal library (Gagné et al. 2015; Robert et al. 2016) to try to narrow down the spectral type from K-band spectroscopy only. With a reduced χ2 of 2.4, the best ﬁt indicates a spectral type close to L7 BD, L11, page 4 of 6 L11, page 4 of 6 GRAVITY Collaboration: Astrometry and K-band spectroscopy of HR 8799 e GRAVITY Collaboration: Astrometry and K-band spectroscopy of HR 8799 e GRAVITY Collaboration: Astrometry and K-band spectroscopy of HR 8799 e in agreement with Bonnefoy et al. (2016) and Greenbaum et al. (2018). A T-type BD spectrum is clearly ruled out. The reduced χ2 increases to 3 for spectral types ≈L4, which is signiﬁcant with 230 ◦of freedom. Similarly, we ﬁtted a grid of BT-Settl 2014 synthetic spectra (Baraﬀe et al. 2015) to derive a temperature and a surface density. The best ﬁt was obtained for a temperature of 1400 K and a surface gravity of 104 cm s2. This corresponds to a planetary radius of 0.8 RJup. Lisbon and Porto, and the European Southern Observatory. Part of this work was supported by the European Union under ERC grant 639248 LITHIUM. J.W. is supported by the Heising-Simons Foundation 51 Pegasi b postdoc- toral fellowship. A.A., P.G., and N.A. acknowledge funding from Fundação para a Ciência e Tecnologia through grants PTDC/CTE-AST/116561/2010, SFRH/BD/52066/2012, COMPETE FCOMP-01-0124-FEDER-019965, UID/ FIS/00099/2013, and SFRH/BSAB/142940/2018. R.G.L. acknowledges funding by H2020 Marie Curie fellowship 706320. Lisbon and Porto, and the European Southern Observatory. 6 Universidade de Lisboa – Faculdade de Ciências, Campo Grande, 1749-016 Lisboa, Portugal 2 Max Planck Institute for extraterrestrial Physics, Giessenbach- straße 1, 85748 Garching, Germany 5. Summary and conclusions 2011, ApJ, 743, L16 Pfuhl, O., Haug, M., Eisenhauer, F., et al. 2014, Optical and Infrared Interferometry IV, 9146, 914623 y , , Rameau, J., Chauvin, G., Lagrange, A. M., et al. 2015, A&A, 581, A80 Robert, J., Gagné, J., Artigau, E., et al. 2016, ApJ, 830, 144 Schweitzer, A., Gizis, J. E., Hauschildt, P. H., et al. 2002, ApJ, 566, 435 Snellen, I. A. G., Brandl, B. R., de Kok, R. J., et al. 2014, Nature, 509, 63 Vousden, W. D., Farr, W. M., & Mandel, I. 2016, MNRAS, 455, 1919 Wang, J. J., Graham, J. R., Dawson, R., et al 2018a, AJ, 156, 192 Wang, J., Mawet, D., Fortney, J. J., et al. 2018b, AJ, 156, 272 Zurlo, A., Vigan, A., Galicher, R., et al. 2016, A&A, 587, A57 Rameau, J., Chauvin, G., Lagrange, A. M., et al. 2015, A&A, 581, A80 Robert, J., Gagné, J., Artigau, E., et al. 2016, ApJ, 830, 144 p The interferometric technique brings unique possibilities to characterize exoplanets. With the technique described here, any planet with Kmag ⪅19, ∆Kmag ⪅11, and separation ⪆100 mas is, in theory, observable with GRAVITY. The num- bers are still to be reﬁned, but it would mean that GRAVITY could observe most of the known imaged planets, and maybe in the near future planets detected by radial velocity. Futher- more, the good normalization of the continuum spectrum oﬀers new ways to measure the column density of molecules without the need for smoothing and cross-correlation (e.g., Snellen et al. 2014; Konopacky et al. 2013). Finally, the idea that an interfer- ometer can resolve the surface of exoplanets, giving radius and resolving clouds patchiness, is now becoming more plausible. However, it would require an interferometer with baselines on the order of 10 km. This could be a goal for ESO after ELT construction. 1 LESIA, Observatoire de Paris, Université PSL, CNRS, Sorbonne Université, Univ. Paris Diderot, Sorbonne Paris Cité, 5 place Jules Janssen, 92195 Meudon, France e-mail: sylvestre.lacour@obspm.fr, mathias.nowak@obs pm.fr 2 Max Planck Institute for extraterrestrial Physics, Giessenbach- straße 1, 85748 Garching, Germany 3 3 Max Planck Institute for Astronomy, Königstuhl 17, 69117 Heidel- berg, Germany 4 4 1st Institute of Physics, University of Cologne, Zülpicher Straße 77, 50937 Cologne, Germany 5 5 Univ. References p This radius being incompatible with evolutionary mod- els, we turned to the Exo-REM model (Baudino et al. 2015; Charnay et al. 2018). We found that values of 1150 ± 50 K and log(g) = 4.3 ± 0.3 (error bars 3σ) correctly reﬂect the spectrum in the K band (Fig. 4). According to the luminosity estimated by the model, it corresponds to a radius of R = 1.17+0.13 −0.11 RJup. This gives a model-dependent estimate of the mass of HR 8799 e of 10+7 −4 MJup. Simulations with Exo-REM predict that the LT tran- sition occurs at a lower eﬀective temperature for exoplanets than for ﬁeld brown dwarfs, due to eﬀects of pressure on the forma- tion of iron and silicate clouds. This trend is apparent in Fig. 4, where the LT transition corresponds to a sudden increase in χ2 and occurs at an eﬀective temperature just 100 K lower than our best ﬁt. Allard, F., Homeier, D., Freytag, B., & Sharp, C. M. 2012, EAS Pub. Ser., 57, 3 Baines, E. K., White, R. J., Huber, D., et al. 2012, ApJ, 761, 57 Baraﬀe, I., Homeier, D., Allard, F., & Chabrier, G. 2015, A&A, 577, A42 Baudino, J.-L., Bézard, B., Boccaletti, A., et al. 2015, A&A, 582, A83 Blunt, S., Ngo, H., Wang, J., et al. 2019, Sblunt/Orbitize: Expand Default Orbit Plots (Zenodo) Bonnefoy, M., Zurlo, A., Baudino, J. L., et al. 2016, A&A, 587, A58 Charnay, B., Bézard, B., Baudino, J.-L., et al. 2018, ApJ, 854, 172 Crossﬁeld, I. J. 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Summary and conclusions Grenoble Alpes, CNRS, IPAG, 38000 Grenoble, Fr 6 6 Universidade de Lisboa – Faculdade de Ciências, Campo Grande, 1749-016 Lisboa, Portugal L11, page 5 of 6 L11, page 5 of 6 A&A 623, L11 (2019) 15 Dublin Institute for Advanced Studies, 31 Fitzwilliam Place, Dublin 2, Ireland 7 Faculdade de Engenharia, Universidade do Porto, rua Dr. Roberto Frias, 4200-465 Porto, Portugal g 8 European Southern Observatory, Karl-Schwarzschild-Straße 2, 85748 Garching, Germany 16 Department of Astronomy, University of Michigan, Ann Arbor, MI 48109, USA 17 European Space Agency, Space Telescope Science Institute, 3700 San Martin Drive, Baltimore, MD 21218, USA 9 European Southern Observatory, Casilla 19001, Santiago 19, Chile 10 Observatoire de Genève, Université de Genève, 51 Ch. des Mail- lettes, 1290 Versoix, Switzerland 18 Space Telescope Science Institute, Baltimore, MD 21218, USA , , 11 Max Planck Institute for Radio Astronomy, Auf dem Hügel 69, 53121 Bonn, Germany 19 STAR Institute, Université de Liège, Allée du Six Août 19c, 4000 Liège, Belgium y 12 Sterrewacht Leiden, Leiden University, Postbus 9513, 2300 RA Lei- den, The Netherlands 20 Department of Astronomy, California Institute of Technology, Pasadena, CA 91125, USA , 13 Departments of Physics and Astronomy, Le Conte Hall, University of California, Berkeley, CA 94720, USA 21 Instituto de Astronomía, Universidad Nacional Autónoma de Méx- ico, Apdo. Postal 70264, Ciudad de México 04510, Mexico y 14 CENTRA – Centro de Astrofísica e Gravitação, IST, Universidade de Lisboa, 1049-001 Lisboa, Portugal 22 School of Physics, Astrophysics Group, University of Exeter, Stocker Road, Exeter EX4 4QL, UK L11, page 6 of 6
https://openalex.org/W4210387363	https://discovery.ucl.ac.uk/id/eprint/10162755/1/Medium%20and%20long-term%20efficacy%20of%20psychoeducational%20family%20intervention%20for%20bipolar%20I%20disorder%20Results%20from%20a%20real-world%2C%20mul.pdf	English	null	Medium and long‐term efficacy of psychoeducational family intervention for bipolar I disorder: Results from a real‐world, multicentric study	Bipolar disorders	2,022	cc-by	8,089	This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Bipolar Disorders published by John Wiley & Sons Ltd. DOI: 10.1111/bdi.13182 DOI: 10.1111/bdi.13182 O R I G I N A L A R T I C L E Bipolar Disorders. 2022;24:647–657. Medium and long-term efficacy of psychoeducational family intervention for bipolar I disorder: Results from a real-world, multicentric study Mario Luciano1 \| Gaia Sampogna1 \| Valeria Del Vecchio1 \| Vincenzo Giallonardo1 \| Arcangelo Di Cerbo1 \| Carmela Palummo1 \| Claudio Malangone2 \| Debora Lampis3 \| Franco Veltro4 \| Francesco Bardicchia5 \| Giusy Ciampini6 \| Emanuele Orlandi7 \| Annamaria Moroni8 \| Silvia Biondi9 \| Massimiliano Piselli10 \| Giulia Menculini11 \| Giuseppe Nicolò12 \| Enrico Pompili12 \| Giuseppe Carrà13 \| Andrea Fiorillo1 1Department of Psychiatry, University of Campania “Luigi Vanvitelli”, Naples, Italy 2Mental Health Centre of Ravello, Ravello, Italy 3Mental Health Centre of Lanusei, Lanusei, Italy 4Mental Health Department of Campobasso, Campobasso, Italy 5Mental Health Centre of Grosseto, Grosseto, Italy 6Mental Health Centre of Lanciano, Lanciano, Italy 7Mental Health Centre of Sassuolo, Sassuolo, Italy 8Department of Psychiatry, Niguarda Hospital, Milan, Italy 9Mental Health Centre of Montecatini, Montecatini, Italy 10Mental Health Centre of Foligno, Foligno, Italy 11Department of Psychiatry, University of Perugia, Perugia, Italy 12Mental Health Centre of Colleferro, Colleferro, Italy 13Department of Medicine and Surgery, University of Milano Bicocca, Monza, Italy 1Department of Psychiatry, University of Campania “Luigi Vanvitelli”, Naples, Italy 2Mental Health Centre of Ravello, Ravello, Italy 3Mental Health Centre of Lanusei, Lanusei, Italy 4Mental Health Department of Campobasso, Campobasso, Italy 5Mental Health Centre of Grosseto, Grosseto, Italy 6Mental Health Centre of Lanciano, Lanciano, Italy 7Mental Health Centre of Sassuolo, Sassuolo, Italy 8Department of Psychiatry, Niguarda Hospital, Milan, Italy 9Mental Health Centre of Montecatini, Montecatini, Italy 10Mental Health Centre of Foligno, Foligno, Italy 11Department of Psychiatry, University of Perugia, Perugia, Italy 12Mental Health Centre of Colleferro, Colleferro, Italy 13Department of Medicine and Surgery, University of Milano Bicocca, Monza, Italy Correspondence Mario Luciano, Department of Psychiatry, University of Campania “L. Vanvitelli”, Largo Madonna delle Grazie 1, 80138, Naples, Italy. Email: mario.luciano@unicampania.it Funding information Italian Ministry of Health 1 \| INTRODUCTION interventions (PFI) are recommended by the most updated treat- ment guidelines for bipolar disorder, as adjunctive intervention to pharmacological treatment13,14 for increasing patients’ and relatives’ knowledge about the illness, improving patients’ adherence to treat- ment, and reducing hospitalizations and recurrences.15 However, while most of available studies have explored the short-term effi- cacy of these interventions, only a few studies have explored the medium and long-term impact of PFI on clinical, social and family outcome of patients with bipolar I disorder. Moreover, most studies have been carried out in tertiary settings, with strict selection crite- ria and rigorous methodologies,6 being not generalizable to the real life of patients with BD and their family members. 1 Bipolar disorder (BD) is a chronic psychiatric illness, characterized by an alternance of depressive and manic or hypomanic episodes. In addition to mood instability, BD is associated with significant func- tional impairment, low quality of life, and high suicide rate.1,2 Up to 50% of individuals with bipolar I disorder do not recover from severe manic episodes within one year, and only about 25% return to their previous level of functioning.3 While psychotropic medications re- main the mainstream treatment for bipolar disorder,4 pharmacother- apy alone allows remission only to a minority of patients.5 BD is associated with severe levels of burden on family members and on the society at large due to recurrent mood episodes, frequent hospitalizations, and loss of productivity.3 In particular, several stud- ies found high levels of burden, expressed emotions,6,7 dysfunc- tional coping strategies and significant morbidity8 in the majority of relatives. In fact, taking care of a person with BD is very a demanding task characterized by a sense of powerlessness, hopelessness, and inability to positively cope with the situation.9 This study, funded by the Italian Ministry of Health and coordi- nated by Department of Psychiatry of the University of Campania “L. Vanvitelli”, has been carried out in 11 randomly selected Italian mental health centres. The primary aim of our study was to explore the efficacy of the Falloon psychoeducational intervention16 in patients with bipolar I disorder and relatives as add-on therapy to treatment as usual (TAU) compared to TAU alone in terms of: (1) im- provement of patients’ global psychopathological status and global functioning; (2) reduction of relatives’ objective and subjective burden and improvement of coping strategies. Secondary analyses allowed us to evaluate the medium and long-term efficacy of this intervention. 1 \| INTRODUCTION In this paper we report the results of our study at 1 and 5 years after the intervention. High levels of family burden are associated with poor patients’ outcome, significant morbidity and mortality,10 poor treatment ad- herence and frequent relapses and hospitalizations.3 Moreover, pa- tients living in families with high levels of burden have a reduced social functioning,6 a poor quality of life11 and present residual symptoms more frequently than patients living in families with low levels of family burden.12 Moreover, high levels of family burden are associated also with significant distress in relatives, who frequently show sleep distur- bances, mild mental disturbances, depressive symptoms, psycho- social impairment, emotional exhaustion and a high utilization of mental and physical health care services.10 Abstract Objectives: This study aims to explore the long-term efficacy of a psychoeducational family intervention (PFI) in bipolar I disorder at one and five years post-intervention in terms of improvement of: (1) patients’ symptoms and global functioning and (2) rela- tives’ objective and subjective burden and coping strategies. Methods: This is a multicentre, real-world, controlled, outpatient trial. Recruited pa- tients and key-relatives were consecutively allocated to the experimental interven- tion or treatment as usual. Patients were assessed at baseline, and after one and five years. Results: One hundred and thirty-seventh number families have been recruited; 70 have been allocated to the experimental intervention, and 67 have been allocated to the control group. We observed an increasing positive effect of the PFI on patients’ clinical status, global functioning and objective and subjective burden after one year. wileyonlinelibrary.com/journal/bdi Bipolar Disorders. 2022;24:647–657. 648 \| 648 LUCIANO et al. We also found a reduction in the levels of relatives’ objective and subjective burden and a significant improvement in the levels of perceived professional support and of coping strategies. The efficacy of PFI on patients’ clinical status was maintained at five years from the end of the intervention, in terms of relapses, hospitalizations and suicide attempts. Conclusions: The study showed that the provision of PFI in real-world settings is as- sociated with a significant improvement of patients’ and relatives’ mental health and psychosocial functioning in the long term. We found that the clinical efficacy of the intervention, in terms of reduction of patients’ relapses, hospitalization and suicide attempts, persists after 5 years. It is advisable that PFI is provided to patients with BD I in routine practice. K E Y W O R D S bipolar I disorder, family, long-term efficacy, psychoeducation, real-world 2 \| MATERIALS AND METHODS This is a multicentric real-world controlled study, which included the following phases: (1) randomly selection of 11 Italian mental health centres, stratified by geographical area and population density; (2) development of educational materials and selection of assessment instruments; (3) training of at least 2 mental health professionals in Despite the evidence of a massive involvement of relatives in the health care of patients with BD, family supportive interventions are provided only rarely. In particular, psychoeducational family LUCIANO et al. 649 each participating centre on PFI and on assessment tools; (4) recruit- ment of at least 16 families of patients with bipolar I disorder in each centre. level, occupational status, pharmacological and psychosocial interventions. The Brief Psychiatric Rating Scale (BPRS),17 a semi-structured interview consisting of 24-items, scoring from 1 (none) to 7 (very se- vere), grouped in four subscales: positive symptoms, negative symp- toms, depressive symptoms and manic-hostility symptoms, has been used to assess patients’ clinical status. Patients referring to the outpatient units of participating men- tal health centres have been invited to participate if they were: (a) aged between 18–65 years; (b) in charge to the local mental health centre for at least 6 months, with at least one access per month; (c) experiencing an affective episode in the previous three years; (d) living with at least one adult relative aged 18–70 years; (e) able to provide written informed consent. Patients who were not clin- ically stable at recruitment and those suffering from a severe and disabling chronic physical condition requiring intensive medical care were excluded. The Disability Assessment Schedule (DAS),18 which includes 11 areas (“self-care”, “participation in daily activities”, “slowness”, “so- cial withdrawal”, “family participation”, “affective and marital role”, “parental role”, “social contacts”, “occupational role”, “interest and information”, and “behavior in emergency situations”), has been used to assess patients’ social and personal functioning. A total score as- sessing patients’ overall general functioning is included at the end of the interview. Higher scores indicate a worse social functioning; each area of functioning ranges from 1 (excellent functioning) to 6 (very severe dysfunction). All patients who agreed to participate were asked permission to contact and involve their key-relative(s). For each patient, one or more key-relative could be recruited. Key-relatives were defined as those spending the highest number of hours in contact with the pa- tient during the last year. 2.1 In each participating center, two mental health professionals (one of them being a psychiatrist) received a training course on the in- tervention and on the study protocol. The training consisted of three-monthly sessions, each lasting two and half days (20 h per session). Five supervision meetings lasting one and half days were provided in order to support mental health professionals during the study period. An additional training course on the use of as- sessment tools and to test participants’ inter-rater reliability has been carried out. Adherence to pharmacological treatment was mandatory to be included in the study. Pharmacological treatment regimen was considered adequate if at least one mood stabilizer or one atypical antipsychotic drug was prescribed, in accordance with the NICE guidelines for the management of bipolar disorder.21 Patients and relatives were assessed at baseline (T0), at the end of the intervention (T1), and after one year from the end of the intervention (T2). After five years (T3), information on the follow- ing course indicators were collected: number of relapses requir- ing a significant modification of the pharmacological treatment, number of hospitalizations and total length of hospitalizations, suicide attempts and number of suicide attempts. In the present paper T2 and T3 data are reported. T1 data have been reported elsewhere.6,8 2 \| MATERIALS AND METHODS The Personal Problems’ Questionnaire (PPQ), a self-reported questionnaire including 34 items, grouped in 7 subscales (subjec- tive and objective burden, practical and affective support, social and professional help, social network) has been used to assess pa- tients’ burden of illness. Each item scores from 1 (“never”) to 4 (“al- ways”)19 The same questionnaire, relatives’ version (Family Problem Questionnaire – FPQ), has been used to assess relatives’ objective and subjective burden.20 Patients and their relatives were consecutively allocated to re- ceive the experimental intervention or the control group. Patients from both groups continued to receive the treatment usually pro- vided in their centre (TAU), which included regular outpatient psy- chiatric assessment, pharmacological treatment and management of medications’ side effects. All patients received an adequate phar- macological treatment according to the NICE guidelines6 for the whole duration of the study. Patients who refused to take medica- tions were excluded. More information on the study methodology is reported elsewhere.6 The study has been carried out in compli- ance with the ethical principles of the Declaration of Helsinki and has been approved by the Ethical Committee of the University of Campania “L. Vanvitelli (number: 9556/2009). The Social Network Questionnaire (SNQ),19 a self-administered questionnaire including 15 items grouped in 4 subscales (practical support, affective support, social and professional help, and help in emergency) has been used to assess patients’ and relatives’ social network. Items range from 1 (“never”) to 4 (“always”). The Family Coping Questionnaire (FCQ),19 a self-administered questionnaire consisting of 34 items, has been used to assess rel- atives’ coping strategies. Items are rated on a 4-level scale, from 1 (“never”) to 4 (“always”), grouped into 11 subscales (seeking for in- formation on patient's illness, positive communication toward the patient, relatives’ maintenance of social interests, patient's involve- ment in social activities, talking with friends about the patient's con- dition, coercion, avoidance, resignation, use of alcohol and drugs, and collusion). 3 \| RESULTS The experimental intervention is based on the psychoeducational family intervention model developed by Falloon16 for patients with schizophrenia and their relatives. The Falloon model has been adapted to bipolar disorder by our research group to be used in Italian non-tertiary settings, taking into account recent changes occurred in families’ composition and structure.22 The approach has been adapted to BD according to the following methodology: (1) analysis of scientific literature, handbooks and manuals on bi- polar disorder23-26; (2) focus groups with relevant stakeholders (researchers, expert clinicians, users and carers) in order to iden- tify the most important components to be included in the inter- vention; (3) development or adaptation of the following sessions: (1) individual and family assessment; (2) information on clinical and course characteristics of BD, its treatment, early warning signs, management of suicidal behaviors; (3) communication skills; (4) problem solving skills. One of the 11 centres did not provide the intervention and was excluded from the study. 143 families of patients with BD from the remaining 10 centres have been invited to par- ticipate. Of these, 137 agreed to participate and have been randomly allocated to the experimental (70 patients and 85 relatives) or the control group (67 patients and 70 relatives). Fourteen families dropped-out during the first six month of the study (10 families from the experimental group and 4 from the control group), due to logistic difficulties in attending the sessions, lack of interest, onset of a severe physical disorder in one family member, illness exacerbation in the patient. The retention rate was 93% in the experimental group and 94% in the control group at T1, with a study sample of 123 families (60 patients and 72 relatives in the experimental group and 63 pa- tients and 67 relatives in the control group). There have been no dropouts at T2. After 5 years (T3), a total of 23 patients (11 from the experimental and 13 from the control group) have not been reassessed due to patients’ death (1 for suicide and 4 for physical illnesses), onset of a severe Alzheimer disease (N = 1), or patients not being in charge anymore to the participating mental health centre (N = 11). Six patients were excluded from the analyses because they refused to take medications (two patients from the experimental and four from the control group). 2.5 \| Statistical analyses Differences between patients’ and relatives’ socio-demographic characteristics from the experimental and the control groups have been tested using χ2 or t-test for independent samples, as appro- priate. Differences at T0 and T2 samples, and at T0 and T3, with respect to patients’ and relatives’ socio-demographic characteris- tics have been explored with χ2 or t-test for independent samples, as appropriate. The impact of the intervention on patients’ social and clinical variables, as well as on relatives’ burden and coping strategies after one year in the two groups, has explored by the Student t-test for paired samples. Linear regression models have been used to test the impact of socio-demographic and clinical characteristics on patients’ clinical status and global functioning at one year. Logistic regression analyses have been carried out to test the efficacy of the experimental intervention on course indi- cators at five years. All clinical and socio-demographic variables 650 \| 2.3 \| Inter-rater reliability LUCIANO et al. 650 that were significantly different at the relevant univariate analy- ses, as well as other potential explanatory variables identified from the literature (i.e., age, gender, years of education, BPRS subscores at baseline, employment and number of relatives per each patient), have been included in the regression model. The T0 BPRS subscales’ scores have been used as independent variables in all regression analyses in order to correct multivariate models for the baseline symptom levels. Data analysis has been carried out using SPSS Statistical software, Version 18.0, with a signifi- cance level of p < 0.05. Cohen's kappa coefficient for BPRS was between 1.0 and 0.90 for 43% of items, between 0.89 and 0.70 for 29%, and between 0.69 and 0.50 for the remaining 28% of items. Cohen kappa coefficient for DAS was between 1 and 0.90 for 39% of items, between 0.89 and 0.70 for 16%, and between 0.69 and 0.50 for the remaining 45% of items. 3 \| RESULTS The retention rate at T3 was 70% in the experimental group and 75% in the control group, with a final study sample of 99 patients (49 in the experimental and 50 in the control group). There were no statistically significant differences at T0 between patients and relatives who completed the T3 as- sessments and those who dropped-outs in terms of sociode- mographic and clinical characteristics. Sessions are provided every 10 days (three times a month) for 4 to 6 months (about 12–18 sessions in total). Each session lasts about 90 min. Site and frequency of sessions are adapted to families’ needs and mental health professionals’ duties and workloads. Leaflets and other written materials are usually given to family members, when- ever relevant. 2.2 \| Assessment instruments Sociodemographic and clinical characteristics of patients and rela- tives have been recorded through ad-hoc schedules. Recorded infor- mation included diagnosis, illness duration, age at onset, number of affective episodes and of previous hospitalizations, age at first hos- pitalization, number of suicide attempts, age, gender, educational 3.2 \| Relatives’ socio-demographic characteristics Relatives’ socio-demographic characteristics are reported in Table 2. At T0 there were no statistically significant differences between treated relatives and those included in the control group. We also observed an improvement of coping strategies of treated relatives. In particular, family members more frequently adopted problem-oriented coping strategies, such as positive communication with the patient (p < 0.01) and seek for information (p < 0.05), and less frequently used emotion-focused strategies, such as collusion (p < 0.0001), resignation (p < 0.001) and avoidance (p < 0.01) (Table 4). In the control group, we found an increase of coercion (p < 0.001), and a reduction of patients’ involvement in social activities (p<.05) and of positive communication with the patients (p < 0.01) (Table 4). 3.3 \| Efficacy of psychoeducational family intervention on patients’ clinical status, global functioning and personal burden at one year We found an increasing positive effect of the intervention on clinical status and global functioning was found in the experimental group. In particular, we observed a significant reduction at the BPRS posi- tive (p < 0.05), negative (p < 0.01) and depression/anxiety symptoms’ subscales (p < 0.001), as well as a significant improvement at the DAS global score (p < 0.01) (Table 3). Moreover, we also found a significant improvement in patients’ objective (p < 0.001) and subjective burden (p<.001). There were no significant changes in the control group with respect to patients’ clinical status, social functioning and personal burden (Table 3). 3.4 \| Efficacy of psychoeducational family intervention on family functioning at one-year (74.9 ± 70.6 months in the experimental group vs. 103.2 ± 73.1 in the control group, p < 0.05) (Table 1). We observed a reduction in the levels of family objective (p < 0.01) and subjective burden (p < 0.001) and a significant improvement in the levels of perceived professional support (p < 0.001) after one year from the end of the intervention in the experimental group. There were no significant differences in the control group (Table 3). \| LUCIANO et al. 651 \| TA B LE 1 Socio-demographic features of the patients at T0 TA B LE 1 Socio-demographic features of the patients at T0 Total sample (N = 137) Experimental group (N = 70) Control group (N = 67) p Gender, F % (n) 62.7 (86) 60.3 (42) 65.7 (44) NS Age, M (sd) 47 (±11.1) 46.3 (10.0) 48.3 (±12.1) NS Marital status, married, yes, % (N) 60.3 (82) 64.3 (45) 56.1 (37) NS Level of education, % (N) NS Primary school degree 47.1 (64) 41.4 (29) 53 (35) High school degree 44.5 (61) 47.1 (33) 39.4(28) University degree 9.6 (13) 11.4 (8) 7.6 (5) Employed, Yes, % (N) 38.3 (55) 42.5 (31) 34.3 (23) NS Number of family members, M (sd) 3.3 (±1.1) 3.4 (±1.0) 3.2 (±1.1) NS Times in charge to MHC, months, M (sd) 88 (±72.8) 74.2 (±70.3) 103.3 (±73.7) <.05 Duration of illness, years, M (sd) 14.6 (±9.5) 13.7 (±9.3) 15.8 (±9.7) NS No. voluntarily admission from onset of the disorder, M (sd) 2.7 (±3.6) 2.6 (±3.8) 2.9 (±3.4) NS No. voluntarily admission during the last year, M (sd) 0.4 (±0.7) 0.4 (±0.6) 0.5 (±0.8) NS No. involuntarily admission from onset of the disorder, M (sd) 1 (±3.1) 0.7 (±1.8) 1.4 (±4.1) NS Involuntarily admission during the last year; yes % (N) 7.1 (10) 5.5 (4) 9 (6) NS Suicide attempts, yes %(N) 23.4 (32) 23.6 (17) 21.7 (15) NS BPRS- positive symptoms; M (sd) 1.2 (±0.4) 1.2 (±0.3) 1.3(±0.4) NS BPRS- negative symptoms; M (sd) 1.4 (±0.5) 1.3 (±0.5) 1.5(±0.5) NS BPRS-manic symptoms; M (sd) 1.3 (±0.5) 1.3 (±0.5) 1.3(±0.5) NS BPRS- depressive symptoms; M (sd) 2 (±0.7) 2.0 (±0.7) 2.0(±0.8) NS DAS- global score; M (sd) 2.9 (±0.9) 2.9 (±0.9) 2.9(±1.0) NS 3.4 \| Efficacy of psychoeducational family intervention on family functioning at one-year 3.1 \| Patients’ socio-demographic and clinical characteristics The socio-demographic characteristics of the global sample and of the two groups are reported in Table 1. At T0 there were no sta- tistically significant differences between the two groups, with the exception of time in charge to the local mental health centre .5 \| Linear regression models The efficacy of the intervention on patients’ symptoms and global functioning has been confirmed by the linear regression models, which explained 31.3% of the total variance for DAS global score, LUCIANO et al. 52 \| 652 TA B LE 2 Socio-demographic characteristics of relatives at T0 Total sample N=155 Experimental group N=85 Control group N=70 p Gender, F % (N) 58.7 (91) 58.3 (49) 60.0 (42) NS Age, M (sd) 51.7 (±13.5) 51.2 (13.7) 52.8 (13.6) NS Marital status, married, yes, % (N) 67.7 (105) 66.7 (56) 70 (49) NS Level of education, % (N) NS Primary school degree 54.4 (84) 53.6 (45) 55.7 (39) High school degree 38.6 (61) 36.9 (33) 40.0 (28) University degree 7 (11) 9.5 (8) 4.3 (3) Employed, Yes, % (N) 49.0 (76) 53.6 (45) 44.3 (31) NS Type of family member, % (N) NS Parent 28.7 (45) 25.3 (21) 32.9 (24) Spouse 48.4 (76) 51.8 (45) 44.3 (31) Son 14 (22) 12.0 (11) 15.5 (11) Sibling 7 (11) 9.6 (8) 4.3 (3) Other 1.9 (3) 1.2 (1) 2.9 (2) Years of cohabiting with patient, M (sd) 25.8 (±12.1) 24.4 (±12.2) 27.6 (±11.8) NS Hours daily spend with patient, M (sd) 6.8 (±3.6) 6.5 (±3.3) 7.2 (±4) NS 55.2% of variance for the BPRS depressive/anxiety subscale, 45.2% of variance for the negative subscale and 23.5% of variance for the positive symptom subscale. Italian population of patients with BDI, given the random selection of participating mental health centres, after stratification for geo- graphic area and population density. At one year, patients receiving the experimental intervention had a better social functioning and lower depressive/anxiety, nega- tive and positive symptoms (Table 5). The efficacy of PFI in bipolar disorder has been investigated mainly in short to medium-term follow-up studies (usually no more than one year), while only two studies have tested its effectiveness after four27 and 5 years,28,29 but in both studies the intervention was provided in group format. 3.6 \| Five-year efficacy of the experimental intervention Although the efficacy of the experimental intervention immedi- ately after the treatment had been already confirmed in our previous analyses,6,8 we found that the intervention significantly improved patients’ positive, negative and depressive/anxiety symptoms, and their global functioning, at the medium-term follow-up (Figure 1). These results, which are largely overlapping with those found imme- diately after the end of the intervention, highlight that the positive effects of the intervention on patients’ symptoms and global func- tioning are sustained over time, and that this intervention may be useful in stabilizing patients from an illness characterized by mood instability and emotional overreactivity. This medium-term effect may be due to increased adherence to medications,30 a better rec- ognition of early warning signs (with consequent reduction of clinical relapses), improvement of patients’ and relatives’ problem-solving skills, or improvement of the family functioning, which is a well- known risk factor for relapses. The efficacy of the experimental intervention was maintained after five years from the end of the intervention. In particular, we found significant differences between the experimental and the control groups in terms of patients’ relapses (28.3% in the experimental group vs. 44.4% in the control group, p < 0.05), number of hospitalizations (16.7% in the experimental group vs. 30.2% in the control group, p < 0.01) and total number of suicide attempts (0% in the experimen- tal group vs. 9.5% in the control group, p < 0.01) (Table 6). The positive effect of the experimental intervention on the likelihood of having a relapse or hospitalization in the five years following the end of the in- tervention was confirmed by the linear and logistic regression models (Table 7). Regression models were not applicable for suicide attempts, since there were any in the experimental group. One important finding of our study is the significant improvement of patients’ psychosocial functioning after one year from the end of the intervention. While in the long-term the efficacy of PFI has been mainly investigated in terms of relapse prevention and symptom re- duction,2,12 its effects on patients’ psychosocial functioning has been less frequently considered as an outcome measure. Since BD is usu- ally associated with a significant impairment in patients’ autonomy, social, working and family functioning, this finding may be extremely 4 \| DISCUSSION 654 \| TA B LE 4 Efficacy of the intervention on relatives’ coping strategies (N = 155) Experimental group (N = 85) Control group (N = 70) T0 mean (sd) T2 mean (sd) T0 mean (sd) T2 mean (sd) Collusion 2.1 (0.4) 1.9 (0.3)** 2.1 (0.4) 2.1 (0.4) Patients’ involvement in social activities 3.0 (0.7) 3.0 (0.7) 3.1 (0.7) 2.8 (0.7)* Resignation 2.1 (0.9) 1.6 (0.7)* 1.9 (1.0) 2.0 (1.0) Avoidance 1.4 (0.7) 1.2 (0.4) 1.3 (0.6) 1.4 (0.7) Coercion 2.0 (0.6) 1.9 (0.4) 2.0 (0.6) 2.3 (0.5)* Relatives’ maintenance of social interests 2.7 (0.7) 3.0 (0.7)* 2.5 (0.8) 2.4 (0.8) Positive communication 3.1 (0.6) 3.3 (0.5) 3.1 (0.6) 2.9 (0.6) Seek for information 2.4 (1.0) 2.7 (0.9)** 2.3 (0.9) 1.9 (0.9) Use of alcohol and drugs 1.0 (0.3) 1.1 (0.4) 1.2 (0.6) 1.1 (0.5) Talking with friends about patient's 2.1 (0.9) 1.9 (0.9) 1.9 (1.0) 1.9 (0.9) Note: Abbreviation: sd, standard deviation. p < 0.05; p < 0.01; *p < 0.001. 4 \| DISCUSSION r regression models to test the efficacy of the intervention on patients’ global functioning and levels of psych TA B LE 5 Linear regression models to test the efficacy of the intervention on patients’ global functioning and levels of psychopathology after one year odels to test the efficacy of the intervention on patients’ global functioning and levels of psychopathology TA B LE 5 Linear regression models to test the efficacy of the intervention on patients’ global functioning a after one year TA B LE 5 Linear regression models to test the efficacy of the intervention on patients’ global functioning and levels of psychopathology after one year DAS Depressive Anxiety symptoms Negative symptoms Positive symptoms Number of subjects included in the analysis 123 123 123 123 F (df) 6.49 (10) 11.05 (10) 14.824 (10) 4.52 (10) P <0.0001 <0.0001 <0.000 <0.000 Adjusted R square 0.317 0.460 0.539 0.230 Constant 1.15 (0.33 to 3.071) 0.52 (−0.10 to 1.13) 0.45 (0.04 to 0.85) 0.53 (0.06 to 1.00) B (95% Cis) B (95% CIs) B (95% CIs) B (95% CIs) Experimental treatment −0.35 (−0.65 to −0.05) −0.24 (−0.45 to −0.03)* −0.14 (−0.27 to 0.00)* −0.23 (−0.37 to −0.68)** Patient's gender, female 0.25 (−0.05 to 0.56) 0.19 (−0.02 to 0.41) 0.6 (−0.07 to 0.21) −0.15 (−0.01 to 0.31) Patient's age −0.08 (−0.06 to 0.02) 0.03 (−0.01 to 0.01) 0.9 (−0.00 to 0.01) −0.01 (−0.03 to 0.01) Patient's level of education −0.03 (−0.07 to 0.01) −0.02 (−0.05 to 0.06) −0.02 (−0.04 to 0.00)* −0.09 (−0.34 to 0.01) Employment, yes −0.01 (−0.30 to 0.33) −0.14 (−0.36 to 0.08) −0.02 (−0.16 to 0.12) −0.01 (−0.16 to 0.18) Number of key-relatives 0.12 (−0.9 to 0.33) 0.10 (−0.5 to.25) 0.2 (−0.07 to 0.12) 0.03 (−0.09 to 0.14) Month on caseload of mental health center 0.01 (−0.00 to 0.00) 0.00 (−0.00 to 0.00) 0.00 (0.00 to 0.00) 0.00 (−0.00 to 0.00) BPRS-positive symptoms at baseline 0.25 (−0.41 to 0.90)* 0.18 (−0.28 to 0.62) 0.01 (−0.29 to 0.31) 0.67 (0.32 to 1.01)**** BPRS-negative symptoms at baseline 0.47 (0.10 to 0.84)* 0.11 (−0.14 to 0.37) 0.59 (0.42 to 0.76)** −0.02 (−.22 to 0.18) BPRS-depression/anxiety symptoms at baseline 0.30 (−0.79 to 0.53)* 0.49 (0.34 to 0.66)**** 0.13 (0.02 to 0.23)* 0.35 (0.09 to 0.16) BPRS-manic/hostility symptoms at baseline −0.02 (−.48 to 0.45) −0.06 (−0.38 to 0.25) −0.31 (0.24 to 0.17) −0.17 (−0.41 to 0.07) Abbreviations: B, Beta Coefficient; BPRS, Brief Psychiatric Rating Scale; CIs, Confidence Intervals; DAS, Disability Assessment Scale; df, degree of freedom. 4 \| DISCUSSION To our knowledge, this is the first study exploring the efficacy of psychoeducational family intervention in patients with bipolar I dis- order in 2 follow-up periods, at 1 and 5 years after the end of the intervention. Our sample can be considered representative of the LUCIANO et al. 653 E 3 One-year efficacy of the experimental intervention Patients (N = 123) Relatives (N = 155) Experimental intervention (N = 60) Control group (N = 60) Experimental intervention (N = 85) Control group (N = 70) T0 Mean (sd) T2 Mean (sd) t Student T0 Mean (sd) T2 Mean (sd) t Student T0 Mean (sd) T2 Mean (sd) t Student T0 Mean (sd) T2 Mean (sd) t Student lobal score 2.9 (1.0) 2.5 (0.8) 3.0 3.0 (1.0) 3.0 (1.0) −0.293 NA NA NA NA NA NA tive burden 2.1 (0.9) 1.7 (0.6) 3.8* 2.0 (0.8) 1.9 (0.8) 1.2 1.6 (0.7) 1.4 (0.4) 3.5 1.6 (0.7) 1.6 (0.7) 0.5 tive burden 2.3 (0.8) 1.9 (0.6) 4.7* 2.2 (0.8) 2.1 (0.7) 2.2 2.0 (0.6) 1.6 (0.5) 5.6*** 1.9 (0.6) 1.9 (0.7) −1.0 contacts 2.2 (0.6) 2.3 (0.5) −0.5 2.2 (0.5) 2.2 (0.5) −1.1 2.2 (0.4) 2.3 (0.4) −1.2 2.2 (0.4) 2.2 (0.4) 0.3 cal support 2.8 (0.7) 2.8 (0.6) −0.4 2.8 (0.7) 2.9 (0.6) −1.4 2.4 (0.7) 2.6 (0.7) −1.6 2.4 (0.7) 2.6 (0.6) −2.2* ive support 2.4 (0.4) 2.4 (0.4) −0.7 2.4 (0.4) 2.4 (0.4) 0.6 2.5 (0.6) 2.6 (0.6) −1.6 2.4 (0.6) 2.5 (0.7) −1.0 sional help 2.8 (0.4) 2.8 (0.5) 0.6 2.8 (0.5) 2.8 (0.4) 0.1 2.9 (0.6) 3.3 (0.4) −5.9*** 2.9 (0.7) 3 (0.7) −0.3 n emergencies 2.7 (0.5) 2.8 (0.4) −1.6 2.6 (0.4) 2.6 (0.4) −0.1 2.5 (0.3) 2.6 (0.3) −1.7 2.5 (0.3) 2.5 (0.3) −0.3 positive symptoms 1.2 (0.4) 1.1 (0.2) 2.7* 1.3 (0.4) 1.4 (0.6) −1.5 NA NA NA NA NA NA negative symptoms 1.3 (0.5) 1.2 (0.4) 3.6 1.4 (0.5) 1.4 (0.6) −0.0 NA NA NA NA NA NA depression/anxiety mptoms 2.0 (0.7) 1.6 (0.7) 5.0* 2.1 (0.8) 1.9 (0.8) 1.5 NA NA NA NA NA NA manic/hostility mptoms 1.3 (0.5) 1.3 (0.6) 0.6 1.3 (0.5) 1.4 (0.8) −1.128 NA NA NA NA NA NA LUCIANO et al. 4 \| DISCUSSION In order to improve its availability in routine settings, virtual settings should be explored in terms of efficacy and feasibility.38-42 TA B LE 7 Logistic regression models to test the efficacy of the intervention on patients’ relapses and hospitalizations after five years from the end of the intervention Relapses Hospitalizations Nagelkerke R square 0.394 0.317 P <0.0001 <0.0001 constant 7.09 5.84 B (95% CIs) B (95% CIs) Experimental treatment −1.20 (0.12 to 9.21)* −0.99 (0.95 to 7.64)* Patient's gender, female −0.39 (0.25 to 1.88) −0.97 (0.12 to 1.17) Patient's age 0.90 (0.87 to 0.97) −0.07 (−0.88 to −0.99)* Patient's level of education −.06 (−0.07 to 0.66) −0.15 (0.73 to 1.01) Patient's employment 0.93 (0.87 to 7.38) −0.52 (0.19 to 1.89) Month on caseload of mental health center 0.00 (1.00 to 1.02)* 0.00 (1.00 to 1.02) Number of key-relatives 0.07 (0.46 to 2.50) −0.26 (0.28 to 2.11) BPRS-positive symptoms −1.80 (0.16 to 1.66) −2.26 (0.01 to 1.4) BPRS-negative symptoms −1.28 (0.07 to 1.11) −1.24 (0.05 to 1.62) BPRS-depression/ anxiety symptoms 0.70 (0.92 to 2.90) 0.57 (0.73 to 3.78) BPRS-manic/hostility symptoms −0.66 (0.09 to 8.90) 0.63 (0.37 to 9.28) Abbreviations: B, Beta Coefficient; BPRS, Brief Psychiatric Rating Scale; CIs, Confidence Interval. p < 0.05. Although not significant, we also found a reduction in the total length of hospitalization in treated patients compared to patients from the control group (32.2 ± 37.7 vs. 40.8 ± 25.2 days), confirming that providing PFI may be associated with a significant reduction of the costs of bipolar disorder. For the whole duration of the study, all enrolled patients took regularly the prescribed medications according to NICE Guidelines. Patients who discontinued the pharmacological treatment have been excluded from the analyses. Given the small sample size at 5 years, the mediating effect of the different mood stabilizers on PFI has not been assessed. Further studies may help to verify whether the ef- ficacy of family psychoeducation on the course of bipolar disorder may be at least partially mediated by mood stabilizers such as lithium. The study has some limitations, such as the lack of an active con- trol group and the use of different outcome measures for medium- and long-term assessments. However, the study was conceived as a “real-world” study and therefore it was not possible to include another active intervention as comparator besides TAU, nor to include an in-depth assessment after five years. 4 \| DISCUSSION p < 0.05; p < 0. 01; p < 0.001; **p < 0.0001. Abbreviations: B, Beta Coefficient; BPRS, Brief Psychiatric Rating Scale; CIs, Confidence Intervals; DAS, Disability Assessment Scale; df, degree of freedom. p < 0.05; p < 0. 01; p < 0.001; **p < 0.0001. important in clinical practice31 as it suggests the need for an integrated pharmacological and psychosocial approach in BD. of the experimental intervention (Figure 1). Despite the evidence clearly shows the negative impact of family instability and maladap- tive coping strategies on the long-term outcome of bipolar disorder, relatives’ coping strategies have been considered only rarely as a Moreover, the improvement of relatives’ coping strategies has also been considered among the outcome measures of efficacy LUCIANO et al. 655 TA B LE 6 Five-year efficacy of the intervention Experimental group (N = 49) Control group (N = 50) Relapse N (%) 17 (28.3) 28 (44.4) Number of relapse M ± SD 2.5 ± 2.2 2.4 ± 1.7 Hospitalization N (%) 10 (16.7) 19 (30.2) Days of hospitalization M ± SD 32.2 ± 37.7 40.8 ± 35.2 Suicide's attempt N (%) 0 6 (9.5) Note: Abbreviations: M, Mean; SD, Standard Deviation. p < 0.05; p < 0.01. TA B LE 6 Five-year efficacy of the intervention the important role of family members in the recovery from bipolar disorder.34,35 We also found that PFI reduced family burden one year after the end of the intervention. This finding, which is in line with other studies on the impact of PFI on family burden in bipolar disorder36 and schizophrenia,37 may contribute to the better patients’ clinical outcome at 5 years, confirming the need for these patients to live in stable emotional environments at family and social levels. One of the mains strengths of the study is the evaluation of the efficacy of PFI at 5 years. In particular, we observed a signifi- cant reduction in the number of relapses, hospitalizations and sui- cide attempts in treated patients (Figure 1). This finding confirms that psychoeducation should be considered an integral part of the “disease-management training” of bipolar I disorder28,29 and that it should be routinely provided to these patients according to the recovery-oriented model of mental disorders. REFERENCES several positive outcomes for both patients and relatives. In particular, at one year, the intervention was particularly effective in improving pa- tient's clinical status and psychosocial status, as well as relatives’ cop- ing strategies and perceived professional help, and in reducing family objective and subjective burden. Moreover, for the first time our study showed that a six-month single-family psychoeducational intervention is associated with a five-year improvement of several clinical hard in- dicators, including number of patients’ relapses, hospitalizations and suicide attempts, suggesting a possible impact of the intervention in reducing the costs of illness. Moreover, the intervention was well re- ceived by patients and relatives, as demonstrated by the high retention rate. According to our results, it is possible to conclude that the pro- vision of a psychoeducational family intervention in real-world setting is associated with significant and long-lasting positive effects on pa- tients’ and relatives’ mental health and well-being. Strategies should be implemented worldwide in order to provide this intervention in routine settings for patients with bipolar I disorder and their relatives. 1. Kilbourne AM, Cornelius JR, Han X, et al. Burden of general medical conditions among individuals with bipolar disorder. Bipolar Disord. 2004;6:368-373. 1. Kilbourne AM, Cornelius JR, Han X, et al. Burden of general medical conditions among individuals with bipolar disorder. Bipolar Disord. 2004;6:368-373. 2. Novick DM, Swartz HA. Psychosocial interventions for bipolar II disorder. Am J Psychother. 2019;72:47-57. 2. Novick DM, Swartz HA. Psychosocial interventions for bipolar II disorder. Am J Psychother. 2019;72:47-57. 3. Soo SA, Zhang ZW, Khong SJ, et al. Randomized controlled trials of psychoeducation modalities in the management of bipolar disorder: a systematic review. J Clin Psychiatry. 2018;79:17r11750. 4. Steardo L Jr, Luciano M, Sampogna G, et al. Efficacy of the interper- sonal and social rhythm therapy (IPSRT) in patients with bipolar dis- order: results from a real-world, controlled trial. Ann Gen Psychiatry. 2020;19:15. 5. Pallaskorpi S, Suominen K, Ketokivi M, et al. Five-year outcome of bipolar I and II disorders: findings of the Jorvi Bipolar Study. Bipolar Disord. 2015;17:363-374. 6. Fiorillo A, Del Vecchio V, Luciano M, et al. Efficacy of psychoeduca- tional family intervention for bipolar I disorder: a controlled, multi- centric, real-world study. J Affect Disord. 2015;172:291-299. 6. Fiorillo A, Del Vecchio V, Luciano M, et al. Efficacy of psychoeduca- tional family intervention for bipolar I disorder: a controlled, multi- centric, real-world study. J Affect Disord. 2015;172:291-299. 7. CONFLICT OF INTEREST 9. Siddiqui S, Khalid J. Determining the caregivers’ burden in caregivers of patients with mental illness. Pak J Med Sci. 2019;35:1329-1333. 9. Siddiqui S, Khalid J. Determining the caregivers’ burden in caregivers of patients with mental illness. Pak J Med Sci. 2019;35:1329-1333. Authors declare to have no conflict of interests. Authors declare to have no conflict of interests. 10. Perlick DA, Hohenstein JM, Clarkin JF, Kaczynski R, Rosenheck RA. Use of mental health and primary care services by caregivers of patients with bipolar disorder: a preliminary study. Bipolar Disord. 2005;7:126-135. 4 \| DISCUSSION 656 6 \| 5 years after the end of the intervention 12 months after the end of the intervention Baseline Emoon-focused coping strategies Resigna on Avoidance Collusion Family burden Objec ve burden Subjec ve burden*** FI G U R E 1 Effect of the experimental intervention on patients’ and relatives’ outcome measures at 1 and 5 years. p < 0.05; p < 0.01; *p < 0.001 ACKNOWLEDGEMENTS 8. Sampogna G, Luciano M, Del Vecchio V, et al. The effects of psychoeducational family intervention on coping strategies of relatives of patients with bipolar I disorder: results from a con- trolled, real-world, multicentric study. Neuropsychiatr Dis Treat. 2018;14:977-979. Open Access Funding provided by Universita degli Studi della Campania Luigi Vanvitelli within the CRUI-CARE Agreement. REFERENCES Fiorillo A, Del Vecchio V, Luciano M, et al. Feasibility of a psychoed- ucational family intervention for people with bipolar I disorder and their relatives: results from an Italian real-world multicentre study. J Affect Disord. 2016;190:657-662. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available on re- quest from the corresponding author. The data are not publicly avail- able due to privacy or ethical restrictions. 11. Pompili M, Innamorati M, Gonda X, et al. Affective temperaments and hopelessness as predictors of health and social functioning in mood disorder patients: a prospective follow-up study. J Affect Disord. 2013;150:216-222. 4 \| DISCUSSION We decided to select a limited number of hard clinical indicators in order to collect information on as many patients as possible minimizing the drop-out rate. The reduced number of patients who have been reassessed at 5 years (99 at T3 vs 137 at T0) is another possible limitation of the study. However, we found no statistically significant differences in patients’ and relatives’ socio-demographic and clinical characteristics between T0 and T3 as- sessments. Finally, one more possible limitation is the use of the BPRS to assess affective symptoms rather than more specific instruments for bipolar disorder. This choice was due to the fact that the BPRS is a well-known instrument frequently used in ordinary psychiatric set- tings, and it can be easily used by mental health professionals with different background and after a brief training.43 proxy outcome measure.32,33 When relatives adopt positive coping strategies, patients feel less stigmatized and stressed, and report a reduced rate of relapses and hospitalizations. In our study, we found a significant improvement of relatives’ coping strategies in the group receiving PFI, and increasing levels of maladaptive coping strate- gies (including coercion) in non-treated relatives. The high levels of maladaptive coping strategies in the control group may explain the worse clinical outcome found at five years in this group, confirming The present study gave us the opportunity to assess the efficacy of a single-family psychoeducational intervention in real-world settings over a medium- and long-term period. According to our findings, the provision of psychoeducational family intervention is associated with 656 \| LUCIANO et al. FI G U R E 1 Effect of the experimental intervention on patients’ and relatives’ outcome measures at 1 and 5 years. p < 0.05; p < 0.01; p < 0.001 Clinical indexes Number of relapses Number of hospitaliza ons* Patients Relatives 12 months after the end of the intervention Baseline 5 years after the end of the intervention Family burden Objec ve burden Subjec ve burden* Emoon-focused coping strategies Resigna on* Avoidance Collusion Received support Professional support* Problem-oriented coping strategies Posi ve communica on with the pa ent Seeking for informa on on the illness Clinical symptomatology Posi ve symptoms** Depressive/anxiety symptoms* Nega ve symptoms* Personal funconing Global func oning* 656 \| LUCIANO et al. doi:10.1111/bdi.12609 15. National Institute for Health and Care Excellence (NICE). Bipolar disorder: assessment and management. Last update: February 2020. Available at: https://www.nice.org.uk/guidance/cg185 33. Perlick DA, Miklowitz DJ, Lopez N, et al. Family-focused treatment for caregivers of patients with bipolar disorder. Bipolar Disord. 2010;12:627-637. 16. Falloon IRH. Family management of schizophrenia: a controlled study of clinical, social, family and economic benefits. John Hopkins University Press, 1985. 34. Masten AS. Resilience from a developmental systems perspective. World Psychiatry. 2019;18:101-102. 17. Morosini PL, Casacchia M. Traduzione italiana della Brief Psychiatric Rating Scale, versione 4.0 ampliata (BPRS 4.0). Rivista di riabilitazi- one Psichiatrica e Psicosociale. 1995;3:199-228. 35. Reynolds CF 3rd. Building resilience through psychotherapy. World Psychiatry. 2019;18:289-291. 18. Morosini P. Guida alla valutazione del funzionamento personale e sociale. In: Andrews G, Hunt C, Jarry M, Morosini P, Roncone R, Tibaldi G, eds. Disturbi mentali. Competenze di base, strumenti e tecniche per tutti gli operatori. Centro Scientifico Editore; 2004:272- 277.ISBN: 88-7640-692-1 36. Miziou S, Tsitsipa E, Moysidou S, et al. Psychosocial treatment and interventions for bipolar disorder: a systematic review. Ann Gen Psychiatry. 2015;7(14):19. 37. Koutra K, Triliva S, Roumeliotaki T, et al. Impaired family function- ing in psychosis and its relevance to relapse: a two-year follow-up study. Compr Psychiatry. 2015;62:1-12. 19. Magliano L, Fiorillo A, Malangone C, De Rosa C, Maj M. Family in- tervention working group implementing psychoeducational inter- ventions in Italy for patients with schizophrenia and their families. Psychiatr Serv. 2006;57:266-269. 38. Hickie IB. The role of new technologies in monitoring the evolu- tion of psychopathology and providing measurement-based care in young people. World Psychiatry. 2020;19:38-39. 20. Morosini PL, Roncone R, Veltro F, Palomba U, Casacchia M. Routine assessment tool in psychiatry: the questionnaire of family attitudes and burden. Ital J Psychiatry Behav Sci. 1991;1:95-101. 39. Cohen AS, Schwartz E, Le T, et al. Validating digital phenotyping technologies for clinical use: the critical importance of "resolution". World Psychiatry. 2020;19:114-115. 40. Torous J, Bucci S, Bell IH, et al. The growing field of digital psychia- try: current evidence and the future of apps, social media, chatbots, and virtual reality. World Psychiatry. 2021;20:318-335. 21. National Institute for Health and Care Excellence (NICE) Bipolar disorder: assessment and management. 2014. Available from: https://www.nice.org.uk/guidance/cg185/chapter/1-Recommenda tions#care-for-adults-children-and-young-people-across-all-phase s-of-bipolar-disorder-2 41. Torous J, Choudhury T, Barnett I, Keshavan M, Kane J. Smartphone relapse prediction in serious mental illness: a pathway towards per- sonalized preventive care. World Psychiatry. 2020;19:308-309. 22. ORCID Mario Luciano https://orcid.org/0000-0002-4338-1371 Giuseppe Carrà https://orcid.org/0000-0002-6877-6169 12. Miklowitz DJ, Chung B. Family-focused therapy for bipolar disorder: reflections on 30 years of research. Fam Process. 2016;55:483-499. 12. Miklowitz DJ, Chung B. Family-focused therapy for bipolar disorder: reflections on 30 years of research. Fam Process. 2016;55:483-499. 12. Miklowitz DJ, Chung B. Family-focused therapy for bipolar disorder: reflections on 30 years of research. Fam Process. 2016;55:483-499. LUCIANO et al. 657 13. Salcedo S, Gold AK, Sheikh S, et al. Empirically supported psycho- social interventions for bipolar disorder: current state of the re- search. J Affect Disord. 2016;201:203-214. 30. Hayes SC, Hofmann SG. "Third-wave" cognitive and behavioral therapies and the emergence of a process-based approach to inter- vention in psychiatry. World Psychiatry. 2021;20:363-375. 14. Yatham LN, Kennedy SH, Parikh SV, et al. Canadian Network for Mood and Anxiety Treatments (CANMAT) and International Society for Bipolar Disorders (ISBD) 2018 guidelines for the management of patients with bipolar disorder. Bipolar Disord. 2018;20(2):97-170. doi:10.1111/bdi.12609 31. Post RM, Altshuler LL, Kupka R, et al. 25 years of the International bipolar collaborative network (BCN). Int J Bipolar Disord. 2021;9(1):13. 32. Goossens PJ, Van Wijngaarden B, Knoppert-van Der Klein EA, Van Achterberg T. Family caregiving in bipolar disorder: caregiver con- sequences, caregiver coping styles, and caregiver distress. Int J Soc Psychiatry. 2008;54:303-316. doi:10.1111/bdi.12609 Luciano M, Sampogna G, Del Vecchio V, et al. The family in Italy: cultural changes and implications for treatment. Int Rev Psychiatry. 2012;24:149-156. 42. Aboujaoude E, Gega L, Saltarelli AJ. The retention challenge in re- mote therapy and learning seen through the lens of the COVID-19 pandemic. World Psychiatry. 2021;20:138-139. 23. Goldberg JF, Harrow M. Bipolar Disorders. American Psychiatric Press; 1998. 43. Roncone R, Ventura J, Impallomeni M, Falloon IR, Morosini PL, Chiaravalle E, Casacchia M. Reliability of an Italian standard- ized and expanded Brief Psychiatric Rating Scale (BPRS 4.9) in raters with high vs. low clinical experience. Acta Psychiatr Scand. 1999;100:229-236. 24. Miklowitz DJ, Goldstein MJ. Bipolar disorder: a family- focused treat- ment approach. Guilford Press; 1997. 25. Jones S, Hayward P, Lam D. Coping with bipolar disorder – a guide to living with manic depression. One word publications; 2002. 26. Colom F, Vieta E. Manual de Psicoeducacìon para el trastorno bipolar. Ars medica; 2004. 27. Buizza C, Candini V, Ferrari C, Ghilardi A, Saviotti FM, Turrina C, Nobili G, Sabaudo M, de Girolamo G. The Long-term effectiveness of psychoeducation for bipolar disorders in mental health services. A 4-year follow-up study. Front Psychiatry. 2019;10:873. How to cite this article: Luciano M, Sampogna G, Del Vecchio V, et al. Medium and long-term efficacy of psychoeducational family intervention for bipolar I disorder: Results from a real-world, multicentric study. Bipolar Disord. 2022;24:647– 657. doi:10.1111/bdi.13182 28. Colom F, Vieta E, Sánchez-Moreno J, et al. Psychoeducation for bipolar II disorder: an exploratory, 5-year outcome subanalysis. J Affect Disord. 2009;112:30-35. 29. Colom F, Vieta E, Sánchez-Moreno J, et al. Group psychoeducation for stabilised bipolar disorders: 5-year outcome of a randomised clinical trial. Br J Psychiatry. 2009;194:260-265.
https://openalex.org/W2899391028	https://www.matec-conferences.org/articles/matecconf/pdf/2018/78/matecconf_balcon2018_05003.pdf	English	null	Determination of p-y curves for offshore piles based on in-situ soil investigations	MATEC web of conferences	2,018	cc-by	4,263	Determination of p-y curves for offshore piles based on in-situ soil investigations 1Gdańsk University of Technology, Faculty of Civil and Environmental Engineeri ul. Narutowicza 11/12, 80-233 Gdańsk, Poland Abstract. Offshore piles are subjected to complex loads with considerable lateral component. The pile-soil response to lateral loads can be described with the p-y method. For a given depth the load–deflection relationship is built to simulate the surrounding soil stiffness. This state-of-art paper presents a brief discussion of determination methods for the p-y curves using a standard approach based on the soil parameters derived from laboratory and in-situ tests or directly from field tests. The basic relationships for both cohesive and cohesionless soils are discussed. The advantage of direct design methods to describe the p-y curve relies in the reduction of necessary laboratory tests. 1 Introduction Nowadays, most of offshore wind farms in Europe are located on waters with depths up to 20 m, and the most popular type of foundation structures are monopiles. These structures are appropriate for water depths up to 35 m. They are made of steel, cylindrical pipes with diameters up to 9 m and wall thickness up to 150 mm [1, 2]. This article focuses on free-headed piles subjected to horizontal loads only (no bending moments). The piles under lateral loads can be divided into three categories: ) - flexible, - rigid, - of intermediate stiffness. The lateral resistance for piles under horizontal loading can be calculated by constructing non-linear load-deflection (p-y) curves. This method is most common and recommended in e.g. the Offshore Standards DNV-OS-J101 [3] or in the API Recommended Practice 2A-WSD [4]. The API p-y formulation is proper for long flexible piles with diameters of up to 1m and the L/D ratio around 30. A pile is considered to be long flexible if it meets the condition [5, 6]: 0 3 l L   with 4 / 1 0 4          K I E l p (1) (1) * Corresponding author: kamila.miedlarz@pg.edu.pl MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 https://doi.org/10.1051/matecconf/201821905003 Determination of p-y curves for offshore piles based on in-situ soil investigations Kamila Międlarz1,, and Lech Bałachowski1 1Gdańsk University of Technology, Faculty of Civil and Environmental Engineering, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland 0l L  0l L  0l L  The cases between the relations from eq. (1) and (2) are intermediate and can be considered either flexible or rigid. The lateral response of rigid piles is still often modelled with the use of p-y curves, as for flexible piles, which may not accurately describe their behavior. For this reason, many studies and field testing have been carried out to develop a method more suitable for monopiles [6]. The p-y curves are used to model the soil stiffness as non-linear springs applied between beam-column elements - representing pile in foundation analysis. A division of methods used to determine p-y curves is presented in this article. In the design of offshore support structures it is vital to perform geotechnical tests: both in-situ (CPT/less often DMT test) and laboratory tests (on undisturbed or evenly disturbed samples) [7]. The aim of the work is to collect and discuss the available methods applied to create p-y curves during the offshore pile modelling. The article is the introduction to further research, applying the presented calculation methods and formulas as an input to create an offshore pile model. The presented review of direct and indirect methods for piles subjected to lateral loads considers mainly flexible piles. The next step of the research intends to modify the given approach and adjust it to the behaviour of rigid monopile. g pp j g p The determination of soil stiffness using CPT and DMT is a starting point for the study of monopile foundation. However, the behaviour mechanism of such a foundation is different from the flexible pile case. Lateral deformations of rigid foundations are more uniform, interaction occurs between the base and shaft mechanism, as the monopiles resembles a block foundation. Additional model tests and numerical analysis are necessary to better analyse this behaviour. Corresponding author: kamila.miedlarz@pg.edu.pl © 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/). MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 https://doi.org/10.1051/matecconf/201821905003 where: l0 transfer length, Ep modulus of the pile material, I pile moment of inertia, K soil stiffness. where: l0 transfer length, Ep modulus of the pile material, I pile moment of inertia, K soil stiffness. l0 transfer length, Ep modulus of the pile material, I pile moment of inertia, The monopiles are relatively short and rigid of 10 times greater diameters and length/diameter (L/D) ratio of values lower than 30. They can be considered short rigid piles if their length satisfies the relationship [5]: 0l L  (2) (2) 2.1 Indirect approach In static loading: In static loading:             u c u u p y y p p 3 1 2 c c y y for y y for     8 8 (5)             u c u u p y y p p 3 1 2 c c y y for y y for     8 8 (5) (5) In cyclic loading and X>XR: In cyclic loading and X>XR:                  c u c c u u y y for p y y for y y p p 3 72 ,0 3 2 3 1 (6)                  c u c c u u y y for p y y for y y p p 3 72 ,0 3 2 3 1 (6) (6) In cyclic loading and X≤XR: In cyclic loading and X≤XR: In cyclic loading and X≤XR: In cyclic loading and X≤XR:                                               c R u c c c c R u c c u y y for X X p y y y for y y y X X p y y for y y p p 15 72 ,0 15 3 12 3 1 1 72 ,0 3 2 3 1 (7)   cy y for 3                                    c R u c c c c R u y y for X X p y y y for y y y X X p p 15 72 ,0 15 3 12 3 1 1 72 ,0 (7) (7) with D y c c     5,2 here: with D y c c     5,2 where: with D y c c     5,2 where: where: εc strain which occurs at one-half the maximum stress in laboratory undrained compression tests. 2.1 Indirect approach The non-linear p-y curves presented in API RP 2A-WSD [4] and DNV CN 30.4 [7] are used to design laterally loaded pile structures with diameters up to 2,5 m. They can be also applied in case of not ideally flexible piles. Construction of p-y curves depends on the soil type and differs for cohesive and cohesionless soils. In the case of cohesionless soils it is necessary to estimate the effective friction angle.. g In the case of cohesive soils the ultimate lateral resistance is based on undrained shear strength (gained from laboratory testing) to be calculated as [4]:              D c X c J D X c p u u u u 9 ) ' 3 (  for for R R X X X X    0 (3) (3) 2 2 rences 219, 05003 (2018) https://doi.org/10.1051/matecconf/201821905003 MATEC Web of Conferences 219, 05003 (2018) https://doi.org/10.1051/matecconf/201821905003 BalCon 2018 MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 https://doi.org/10.1051/matecconf/201821905003 where: X depth below soil surface, XR transition depth, below which the value ((3cu+γ' X)D+JcuX) exceeds 9cu [m], here: X depth below soil surface, XR transition depth, below which the value ((3cu+γ' X)D+JcuX) exceeds 9cu [m], J c D D X u R     ' 6  (4) J c D D X u R     ' 6  (4) where: D pile diameter, p , cu undrained shear strength for undisturbed soil samples, γ' effective unit weight of soil, J empirical coefficient with values from 0,25 to 0,50 (the upper limits value is assumed for soft, normally consolidated cohesive soils). 2.1 Indirect approach where: εc strain which occurs at one-half the maximum stress in laboratory undrained compression tests. where: εc strain which occurs at one-half the maximum stress in laboratory undrained compression tests. In the case of cohesionless soils, p-y curves are also non-linear but can be approximated [4]: [4]:             y p A X k p A p u i init u i tanh (8) (8) 3 https://doi.org/10.1051/matecconf/201821905003 MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 where: Ai factor to account for static or cyclic loading: Ai=(3-0,8(∙z/D))>0,9 for static and Ai=0,9 for cyclic loading, pu static ultimate resistance at depth z, Y lateral deflection, kinit initial modulus of subgrade reaction dependent on the friction angle (see [3]). where: Ai factor to account for static or cyclic loading: Ai=(3-0,8(∙z/D))>0,9 for static and Ai=0,9 for cyclic loading, y g pu static ultimate resistance at depth z, pu static ultimate resistance at depth z, Y lateral deflection, , kinit initial modulus of subgrade reaction dependent on the friction angle (see [3]). kinit initial modulus of subgrade reaction dependent on the friction angle (see [3]). The value of ultimate lateral resistance is soil depth dependent. It has been found that in the case of shallow waters it can be calculated from eq. (9) in the case of deeper waters from eq. (10). At a particular depth, the equation leading to lower value should be considered decisive [4]. X D C X C pus       ' ) ( 2 1  (9) X D C pud     ' 3  (10) (9) (10) where: C1, C2, C3 coefficients dependent on the friction angle ϕ’ (see [3]). where: C1, C2, C3 coefficients dependent on the friction angle ϕ’ (see [3]). The friction angle can be obtained from laboratory tests or in-situ data. If the laboratory data is not available, the values of effective friction angle in sands can be evaluated from CPTU using eq. (11) [8] or eq. 2.1 Indirect approach (12) [9]:                  0 ' log 38 ,0 1,0 arctan ' v c CPT q   (11) ) log( 0, 11 6, 17 ' 1 c CPT q      (12)                  0 ' log 38 ,0 1,0 arctan ' v c CPT q   (11) (11) ) log( 0, 11 6, 17 ' 1 c CPT q      ) log( 0, 11 6, 17 ' 1 c CPT q      (12) (12) where: 5,0 0 1 '             v c c Pa Pa q q  (13) (13) with Pa = 100 kPa, σ’v0 - vertical effective stress [kPa]. with Pa = 100 kPa, σ’v0 - vertical effective stress [kPa]. Safe estimation of effective angle of internal friction in sands can be also determined from DMT [10]: Safe estimation of effective angle of internal friction in sands can be also determined from DMT [10]: D D DMT K K 2 log 1,2 log 6, 14 28 '        (14) (14) In the case of undrained shear strength, the following equations can be used for clays: kt v t CPT u N q c 0 ,    (15) (15) 25 ,1 0 , ) 5,0 ( 22 ,0 D v DMT u K c      (16) (16) where: Nkt cone factor for clays (default to 15); Nkt=10,5+7∙log(Fr) Fr normalized friction ratio, MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 https://doi.org/10.1051/matecconf/201821905003 % 100 ' 0    v t s r q f F  (17) % 100 ' 0    v t s r q f F  (17) KD horizontal stress index; KD=(p0-u0)/σv0, p0 pressure applied to the soil at the start of the expansion, u0 hydrostatic pore water pressure. KD horizontal stress index; KD=(p0-u0)/σv0, p0 pressure applied to the soil at the start of the expansion, u0 hydrostatic pore water pressure. 2.2 Direct approach This approach uses the results of advanced in-situ soil investigations to predict the p-y curve and pile response to lateral loading. It reduces the errors due to inaccurate estimation of undrained shear strength and friction angle with the use of eq. (11),(12),(15), it also eliminates the need for laboratory testing on undisturbed soil samples [11]. Based on API and DNV methods Lehane and Truong [12] determined the construction of p-y curves directly from CPT data. They suggested the following equations for soft clay [12]:   3 0 98 ,3 026 tanh 5,0 85 ,0                            D X for D X D y I p p r u (18) (18)   3 3,2 151 ,0 tanh 85 ,0                    D X for D y I p p r u (19) (19) where: kt n r N q E I       3 1 (20) kt n r N q E I       3 1 (20) kt n r N q E I       3 1 (20) and Ir rigidity index G/cu, G shear modulus (from eq. (22)) qn net cone resistance; qn=qt-σv0, qn cone tip resistance; qt=qc+(1-a)∙u2, a cone coefficient, E Young’s modulus (from eq. (21)). and Ir rigidity index G/cu, G shear modulus (from eq. (22)) qn net cone resistance; qn=qt-σv0, qn cone tip resistance; qt=qc+(1-a)∙u2, a cone coefficient, E Young’s modulus (from eq. (21)). cone tip resistance; qt=qc+(1-a)∙u2, cone coefficient, a cone coefficient, E Young’s modulus (from eq. (21)).   68 ,1 55 ,0 0 10 015 ,0 ) (       cI v tq E  (21) 2) 5, 18 ln 6, 51 ( 8,9    sf G  (22) (21) (22) and p0 and p1 – corrected readings from DMT. and p0 and p1 – corrected readings from DMT. and p0 and p1 – corrected readings from DMT. For cohesionless soils, also the eq. (3) is used, however the ultimate lateral resistance pu is the lower value from eq. (26) (Reese, 1974) [17] and eq. (27) [18]:        tan ' tan ' 0         p a p v u K x K K D p (26)   a p a p v u K K K K D p          ' tan ' tan 2 ' 2 3 0    (27) (26) (27) where: 2 ' 45      (28) 2 ' 45      (28) D F E y D v c        )' sin 1( ' ' sin 17 ,4 0     (29) (29) and 1   F , Ka active earth pressure coefficient, Kp passive earth pressure coefficient. Parameters such as friction angle and undrained shear strength are calculated with the help of equations (14) and (16), applying the data from the DMT test [19]. Regarding sands, Lehane and Suryasentana (2014) [13] proposed:                                            89 , 0 2 ,1 62 ,0 75 ,0 67 ,0 1 4,2 D y D X c e D x X y q D X y p (23) (23) Another method (Robertson, 1989) [14] to obtain the p-y curves for laterally loaded piles is based on DMT data. For cohesive soils, the equation of p-y curves is the same as in Another method (Robertson, 1989) [14] to obtain the p-y curves for laterally loaded piles is based on DMT data. For cohesive soils, the equation of p-y curves is the same as in 5 5 5 https://doi.org/10.1051/matecconf/201821905003 MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 the API Recommendations (eq. (3),(4)), however the equation for the pile deflection changes [15,16] and yc can be evaluated from: the API Recommendations (eq. (3),(4)), however the equation for the pile deflection changes [15,16] and yc can be evaluated from: D c u c E F D c y     5,0 67 , 23 (24) (24) where: Fc empirical factor Fc=10, ED dilatometer modulus. where: Fc empirical factor Fc=10, ED dilatometer modulus. Fc empirical factor Fc=10, Fc empirical factor Fc=10, ED dilatometer modulus. ED dilatometer modulus. ) ( 7, 34 0 1 p p ED    (25) (25) and p0 and p1 – corrected readings from DMT. 3 Monopile lateral stiffness Application of the p-y method, according to the API and DNV guidelines overestimates the subgrade modulus thus produces enormous results if applied to monopiles [1,20]. As a result, many solutions based on both analytical and three-dimensional analysis have been developed [21]. For the purpose of the article, only some of the developed methods are briefly presented. One of the methods, formulated for cohesive soils, was proposed by Dunnavant & O’Neil [22,23]. The main idea of API Recommendation formulas remains the same, however the expression for the static ultimate lateral resistance pu and critical deflection yc changes [24]:              9 , ) ( ' 2 min X D J c X D c p ua v u u  (30) (30) 6 MATEC Web of Conferences 219, 05003 (2018) https://doi.org/10.1051/matecconf/201821905003 BalCon 2018 nces 219, 05003 (2018) https://doi.org/10.1051/matecconf/201821905003 MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 https://doi.org/10.1051/matecconf/201821905003 875 ,0 4 0063 ,0           I E L E y r s c (31) 875 ,0 4 0063 ,0           I E L E y r s c (31) (31) where: cua average shear strength between the layer surface and depth X, Es secant modulus, Lr representative pile length. where: cua average shear strength between the layer surface and depth X, Es secant modulus, where: cua average shear strength between the layer surface and depth X, Es secant modulus, where: cua average shear strength between the layer surface and depth X, Es secant modulus, , representative pile length. , Lr representative pile length.                L D D E I E L s r , 3 , min 286 ,0 4 (32) (32) Similar simplified approach was proposed by Mayne et al. (1992) [25]. These alternative formulations improve the monopile design mainly in overconsolidated clays. For cohesionless soils a new approach was developed by Thieken et al. (2015) [20]. In this method, a deflection-dependent correction factor, is applied to increase the pu value at shallow depths. Calculations are made by means of an iterative procedure. In the first step, deformations are calculated with the use of basic p-y curves. 3 Monopile lateral stiffness Next, the correction factor is applied to the curve and the calculation of deflection is repeated. These steps are reiterated until no significant change is noticed [20]. Apart from analytical approaches, numerical methods are also applied to verify their results [26]. Moreover, model tests in a reduced scale are carried out for a better understanding of the pile – soil interaction. Geotechnical centrifuge is used to investigate the response of model monopiles subjected to different loads [27]. Next, model observations can be extrapolate to analyse the prototype behaviour [28]. Despite many attempts, there is still no specific method for estimating lateral stiffness for large diameter piles. This problem remains unsolved and requires more analysis and systematic research. 4 Conclusions The advantage of direct method to establish p-y curve is the reduction of extent of laboratory tests on high quality samples. The formulations should be however well calibrated and adjusted to given soil conditions. Following direct approach it is impossible to completely reject laboratory testing and the use of correlation between soil strength characteristics and parameters measured by in-situ soil investigation. The well-developed direct and indirect approaches exists for flexible piles up to 2,5 m in diameter. The monopiles used in offshore structures are generally rigid piles, so they cannot be analysed using any standard p-y approach for flexible piles. The case of rigid or intermediate piles needs further efforts considering analytical and numerical solutions combined with advanced physical modelling of centrifuge tests. 2. J. van der Tempel, N.F.B. Diepeveen, D.J. Cerda Salzmann, W.E. De Vries, Wind Power Generation and WInd Turbine Design, 559-594, 768 (2010) 1. M. Achmus, K. Abdel-Rahman, Y. S. Kuo, 11th Baltic Sea Geotechnical Conf. Geotechnics In Maritime Engineering, 1, 463-470 (2008) 2. J. van der Tempel, N.F.B. Diepeveen, D.J. Cerda Salzmann, W.E. De Vries, Wind Power Generation and WInd Turbine Design, 559-594, 768 (2010) References 1. M. Achmus, K. Abdel-Rahman, Y. S. Kuo, 11th Baltic Sea Geotechnical Conf. Geotechnics In Maritime Engineering, 1, 463-470 (2008) 2. J. van der Tempel, N.F.B. Diepeveen, D.J. Cerda Salzmann, W.E. De Vries, Wind Power Generation and WInd Turbine Design, 559-594, 768 (2010) 1. M. Achmus, K. Abdel-Rahman, Y. S. Kuo, 11th Baltic Sea Geotechnical Conf. Geotechnics In Maritime Engineering, 1, 463-470 (2008) 7 MATEC Web of Conferences 219, 05003 (2018) BalCon 2018 https://doi.org/10.1051/matecconf/201821905003 3. Det Norske Veritas, Offshore Standard DNV-OS-J101design of Offshore Wind Turbine Structures, 207-214 (2014) 4. American Petroleum Institute, Recommended Practice for Planning, Designing and Constructing Fixed Offshore Platforms – Working Stress Design, 63-66 (2002) 5. J.L. Briaud, Geotechnical Engineering: Unsaturated and Saturated Soils, 599-603 (2013) 6. F.C. Schroeder, A.S. Merritt, K.W. Sørensen, A. Muir Wood, C.L. Thilsted, D.M. Potts, Frontiers in Offshore Geotechnics III, 735-740 (2015) 7. Det Norske Veritas, Classification Notes No. 30.4 Foundations, 8, 16-20 (1992) 8. P. K. Robertson, R. G. Campanella, Canadian geotechnical journal, 20(4), 718-733 (1983) 9. F. H. Kulhawy, P. W. Mayne, Manual on estimating soil properties for foundation design, EPRI-EL-6800, (1990) 10. G. Totani, S. Marchetti, P. Monaco, M. Calabrese, Intl. Conf. On In situ Measurement of Soil (2001) 11. S. Malhotra, Wind Turbines, 241-247 (2011) 12. P. Truong, B.M. Lehane, 3rd Intl Symp. on Cone Penetration Testing, 975-982 (2014 13. S. Suryasentana, B.M. Lehane, Géotechnique 64, 3, 186-194 (2014) 14. P. K. Robertson, M. P. Davies, R. G. Campanella, Geotechnical Testing Journal, 12(1), 30-38 (1989) 15. J.B. Anderson, F.C. Townsend, B. Grajales, Proc. from the Second Intl. Flat Dilatometer Conf., 50-53 (2006) 16. S. Marchetti, G. Totani, M. Calabrese, P. Monaco, Proc. 4th Intl Conf. DFI-Deep Foundation Institute USA on Piling and Deep Foundations, 1, 263-272 (1991) 17. L. C. Reese, W. R. Cox, F. D. Koop, Offshore Technology in Civil Engineering Hall of Fame Papers from the Early Years, 95-105 (1974) 18. J. M. Murchison, M. W. O'Neill, Analysis and design of pile foundations,174-191 (1984) 19. S. Marchetti, Journal of the geotechnical engineering division, 299-320 (1980) 20. K. Thieken, M. Achmus, K. Lemke, Frontiers in Offshore Geotechnics III , 741-746 (2015) 21. J. Wiemann, K. Lesny, W. Richwien, Proc. 7th German Wind Energy Conf. (DEWEK) (2004) 22. T.W. Dunnavant, M.W. O’Neil, JoGE, 115(1), 95-114 (1989) 23. T.W. Dunnavant, M.W. O’Neil, JoGE, 117(5), 827-829 (1991) 24. M. Arroyo, D. Abadias, M. References Coelho, Frontiers in Offshore Geotechnics III, 693-698 (2015) 25. P.W. Mayne, F.H. Kulhawy, C.H. Trautmann, F. H. Kulhawy, Experimental study of undrained lateral and moment behavior of drilled shafts during static and cyclic loading, EPRI-TR-100221 (1992) 26. M. Achmus, Indian Geotechnical Conference GEOtrendz - 2010, 92-102 (2010) 27. R.T. Klinkvort, M. Poder, P. Truong, V. Zania, Proc. of the 3rd European Conf.on Physical Modelling in Geotechnics (2015) 28. R.T. Klinkvort, O. Hededal, S.M. Springman, IJPMG, 13(2), 38-49 (2013) 8
https://openalex.org/W4378228667	https://journal.stikesyarsimataram.ac.id/index.php/lentera/article/download/200/79	Indonesian	null	PENINGKATAN PEMAHAMAN IBU HAMIL TENTANG PENANGANAN ANEMIA PADA KEHAMILAN DI DESA JEMPONG BARU WILAYAH KERJA PUSKESMAS SEKARBELA KOTA MATARAM	Jurnal Lentera	2,023	cc-by-sa	2,552	LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 Dian Soekmawaty Riezqy Ariendha1 , Irni Setyawati2, Kusniyati Utami3 , Hardaniyati4 dan Yesvi Zulfiana5 1-6Dosen Stikes Yarsi Mataram Email: Diansoekmawaty.ra@gmail.com 1-6Dosen Stikes Yarsi Mataram Email: Diansoekmawaty.ra@gmail.com ABSTRAK Anemia memiliki efek yang sangat buruk, terutama bagi bayi dalam kandungan, termasuk dapat menyebabkan keguguran, bayi lahir tidak cukup bulan, bayi belum berkembang sempurna, pendarahan saat melahirkan, Kontraksi yang tidak teratur, gangguan selama proses persalinan dan bahkan rentan terhadap infeksi mengakibatkan produksi ASI tidak lancar. Pengabdian kepada masyarakat bertujuan untuk meningkatkan pengetahuan tentang pencegahan anemia. Metode: Pelaksanaan kegiatan pengabdian kepada masyarakat dilakukan di kelurahan Jempong. Hasil: ibu hamil yang hadir sangat antusias, setelah mengolah kuesioner sebelum dan sesudah pemberian pendidikan kesehatan, terjadi peningkatan pengetahuan. Kesimpulan: Implementasi memberikan pendidikan kesehatan untuk meningkatkan pengetahuan bagi masyarakat dengan risiko yang mungkin terjadi dengan Kondisi masyarakat sangat perlu dilakukan Selain sebagai penambah dan penambah pengetahuan, juga merupakan tindakan untuk mengurangi risiko masalah kesehatan bagi masyarakat. Kata Kunci: ibu hamil, anemia, penanganan anemia. Kata Kunci: ibu hamil, anemia, penanganan anemia. PENINGKATAN PEMAHAMAN IBU HAMIL TENTANG PENANGANAN ANEMIA PADA KEHAMILAN DI DESA JEMPONG BARU WILAYAH KERJA PUSKESMAS SEKARBELA KOTA MATARAM Dian Soekmawaty Riezqy Ariendha1 , Irni Setyawati2, Kusniyati Utami3 , Hardaniyati4 dan Yesvi Zulfiana5 1-6Dosen Stikes Yarsi Mataram Email: Diansoekmawaty.ra@gmail.com Dian Soekmawaty Riezqy Ariendha1 , Irni Setyawati2, Kusniyati Utami3 , Hardaniyati4 dan Yesvi Zulfiana5 1-6Dosen Stikes Yarsi Mataram Email: Diansoekmawaty.ra@gmail.com Keywords: Pregnancy, anemia, anemia treatment Corresponding Author: Dian Soekmawaty Riezqy Ariendha (email: Diansoekmawaty.ra@gmail.com ), Indonesia. Corresponding Author: Dian Soekmawaty Riezqy Ariendha (email: Diansoekmawaty.ra@gmail.com ), Indonesia. LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 ABSTRACT Anemia has a very negative effect, especially for babies in the womb, including it can cause miscarriage, babies born not quite months, babies are not fully developed, bleeding during childbirth, irregular contractions, disruption during the delivery process and susceptibility to infection can even result in a lack of production of breast milk. Aim of community services is to improve knowledge about prevention of anemia. Methods: The implementation of community service activities is carried. Results: pregnant women who attended were very enthusiastic, after processing the questionnaire before and after the provision of health education, there was an increase in knowledge. Conclusion: Implementation of providing health education to increase knowledge for the community with risks that may occur with the condition of the community is very necessary to be done Apart from being an enhancer and an increase in knowledge, it is also an action to reduce the risk of health problems for the community 226 \|LENTERA (Jurnal Pengabdian) Keywords: Pregnancy, anemia, anemia treatment Keywords: Pregnancy, anemia, anemia treatment 226 \|LENTERA (Jurnal Pengabdian) LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 laporan KIA Dinas Kesehatan Provinsi NTB tahun 2018 terdapat 2,32% ibu hamil mengalami anemia 618 orang ibu hamil, dimana 336 orang (2,9%) diantaranya menderita anemia (Laporan Dinas Kesehatan NTB, 2018).[3] oksigen) tidak cukup untuk memenuhi kebutuhan fisiologis tubuh [10]. Kebutuhan fisiologis spesifik bervariasi dengan kebutuhan seseorang usia, jenis kelamin, ketinggian tempat tinggal di atas permukaan laut (ketinggian), merokok perilaku, dan berbagai tahap kehamilan [11]. Kekurangan zat besi diperkirakan menjadi penyebab paling umum dari anemia secara global, tetapi nutrisi lainnya defisiensi (termasuk folat, vitamin B12 dan vitamin A), akut dan kronis peradangan, infeksi parasit, dan kelainan bawaan atau didapat yang mempengaruhi sintesis hemoglobin, produksi sel darah merah atau sel darah merah kelangsungan hidup, semua dapat menyebabkan anemia[12]. Ibu hamil dikatakan anemia apabila kadar hemoglobin dalam darah kurang dari 11 gram/liter [13]. Menurut Kementerian Kesehatan RI ada 3 penyebab anemia yaitu defisiensi zat gizi, perdarahan (loss of blood volume), dan hemolitik[14]. Gejala yang sering ditemui pada penderita anemia adalah 5 L (Lesu, Letih, Lemah, Lelah, Lalai), disertai sakit kepala dan pusing, mata berkunang- kunang, mudah mengantuk, cepat capai serta sulit konsentrasi. Secara klinis penderita anemia ditandai dengan “pucat” pada muka, kelopak mata, bibir, kulit, kuku dan telapak tangan [15] oksigen) tidak cukup untuk memenuhi kebutuhan fisiologis tubuh [10]. Kebutuhan fisiologis spesifik bervariasi dengan kebutuhan seseorang usia, jenis kelamin, ketinggian tempat tinggal di atas permukaan laut (ketinggian), merokok perilaku, dan berbagai tahap kehamilan [11]. Kekurangan zat besi diperkirakan menjadi penyebab paling umum dari anemia secara global, tetapi nutrisi lainnya defisiensi (termasuk folat, vitamin B12 dan vitamin A), akut dan kronis peradangan, infeksi parasit, dan kelainan bawaan atau didapat yang mempengaruhi sintesis hemoglobin, produksi sel darah merah atau sel darah merah kelangsungan hidup, semua dapat menyebabkan anemia[12]. Ibu hamil dikatakan anemia apabila kadar hemoglobin dalam darah kurang dari 11 gram/liter [13]. Menurut Kementerian Kesehatan RI ada 3 penyebab anemia yaitu defisiensi zat gizi, perdarahan (loss of blood volume), dan hemolitik[14]. Gejala yang sering ditemui pada penderita anemia adalah 5 L (Lesu, Letih, Lemah, Lelah, Lalai), disertai sakit kepala dan pusing, mata berkunang- kunang, mudah mengantuk, cepat capai serta sulit konsentrasi. Secara klinis penderita anemia ditandai dengan “pucat” pada muka, kelopak mata, bibir, kulit, kuku dan telapak tangan [15] Data PWS ibu wilayah puskesmas Karang Pule hingga November 2021 memiliki sasaran ibu hamil sebanyak 1268, dengan angka Persentase di atas menunjukkan bahwa kota Mataram mempunyai wanita yang bermasalah status gizinya meskipun kota Mataram sebagai ibukota provinsi. Kota Mataram telah masuk kategori kota metropolitan sedang yang dilengkapi dengan berbagai fasilitas pelayanan ekonomi, sosial, pendidikan, sarana dan prasarana. Dari sekian banyak fasilitas yang telah ada, idealnya wanita di kota Mataram mempunyai kecukupan asupan nutrisi. Berdasarkan analisis situasi yang telah dijabarkan, masalah yang teridentifikasi diantaranya: 1. Belum adanya kesadaran tentang nutrisi ibu hamil untuk mencegah terjadinya anemia 2. Pengetahuan tentang anemia dan cara pencegahannya 3. Pola konsumsi tablet besi untuk mencegah anemia Analisis Situasi Angka tersebut masih jauh dari target Rancangan Pembangunan Jangka Menengah (RPJM) tahun 2015 sebesar 306 per 100.000 kelahiran hidup dan target Sustainable Development Goals (SDG’s) sebesar 70 per 100.000 kelahiran hidup tahun 2016 [1]. World Health Organization (WHO) melaporkan bahwa prevalensi wanita hamil yang mengalami defesiensi sekitar 37-75% serta semakin meningkat seiring dengan bertambahnya usia kehamilan [7]. Dimana 40% kematian ibu di negara berkembang berkaitan dengan anemia pada kehamilan dan kebanyakan anemia pada kehamilan disebabkan oleh defesiensi besi dan perdarahan akut, bahkan tidak jarang keduanya saling berinteraksi [7] . Sementara persentase wanita hamil dari keluarga miskin terus meningkat (8%) anemia di trimester I, 12% persen, anemia di trimester II dan 29% anemia di trimester III [8] Rencana Pembangunan Kesehatan Jangka Menengah Nasional (RPJMN) pada periode 2015-2019 merupakan. Program Indonesia Sehat dengan sasaran peningkatan derajat kesehatan dan status gizi masyarakat melalui upaya kesehatan dan pemberdayaan masyarakat yang didukung dengan perlindungan finansial dan pemerataan pelayanan kesehatan. Adapun termasuk didalamnya peningkatan kesehatan dan status gizi ibu dan anak [1]. Berdasarkan SDKI 2012 angka kematian ibu di Indonesia sebesar 359 per 100.000 kelahiran hidup [2]. Berdasarkan laporan kabupaten/kota jumlah kasus kematian ibu di Provinsi NTB selama tahun 2016 adalah 92 kasus, menurun dibandingkan tahun 2015 dengan 95 kasus [3]. Adapun kematian ibu di Indonesia masih didominasi oleh tiga penyebab utama kematian, yaitu perdarahan (30.3%), hipertensi dalam kehamilan (27.1%), dan infeksi (7.3%) [4]. Tingginya prevalensi anemia pada ibu hamil merupakan masalah yang tengah dihadapi pemerintah Indonesia saat ini . Data Riset Kesehatan Dasar (RISKESDAS) tahun 2018 menunjukkan terjadinya peningkatan kejadian anemia pada ibu hamil pada rentang waktu tahun 2007 hingga tahun 2018 [4]. Jumlah ibu hamil yang mengalami anemia pada tahun 2007 yaitu sebanyak 24,5%, meningkat di tahun 2013 menjadi 37,1% dan terus mengalami peningkatan hingga 48,9% di tahun 2018 [9] Anemia pada ibu hamil dapat dikatakan “potensial danger to mother and child” (potensial membahayakan ibu dan anak) [5].Oleh karenanya anemia memerlukan perhatian serius dari semua pihak yang ada dalam pelayanan kesehatan. Menurut WHO, tahun 2017 terdapat 40% kematian ibu di Negara berkembang berhubungan dengan anemia kehamilan [6]. Berdasarkan data survey demografi dan kesehatan Indonesia (SDKI) pada tahun 2012 menyebutkan bahwa angka kematian ibu (AKI) di Indonesia sebesar 359 per 100.000 kelahiran hidup [2]. [9] Anemia adalah suatu kondisi di mana jumlah sel darah merah (hemoglobin sebagai pembawa 227 \|LENTERA (Jurnal Pengabdian) LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 Sekarbela Kota Mataram. Solusi yang ditawarkan adalah pemberian pendidikan kesehatan tentang penanganan anemia pada ibu hamil dengan sasarannya adalah ibu hamil. Kegiatan ini terdiri dari beberapa tahap, yaitu pretest, pelaksanaan pendidikan kesehatan, dan posttest. Setelah selesai kegiatan pretest, tim pengabdian memberikan materi pendidikan kesehatan selama 15 menit dan dilanjutkan dengan tanya jawab. Media yang digunakan adalah leaflet dan PPT yang berisi materi dilengkapi gambar sehingga peserta mudah memahami materi yang disampaikan. Para peserta sangat antusias mengikuti kegiatan tersebut, dilihat dari banyaknya peserta yang bertanya dan menjawab ketika diajukan pertanyaan. Pelaksanaan kegiatan dilakukan selama 1 hari, yaitu pemberian pendidikan kesehatan tentang penanganan anemia, yang dilaksanakan pada tanggal 29 Mei 2022 bersamaan dengan kegiatan Posyandu dengan kelompok sasaran adalah ibu hamil. Gambar 2 Penyampaian Materi kepada ibu Hamil METODE Mitra pengabdian kepada masyarakat ini yaitu Kepala Desa Kelurahan Jempong Baru Kecamatan Sekarbela Kota Mataram. Metode pelaksanaan pengabdian ini terdiri dari dua tahap. Pertama merupakan perencanaan kegiatan yang akan dilakukan dan tahap kedua merupakan pelaksanaan kegiatan pengabdian berupa solusi yang telah disetujui kepala Desa Kelurahan Jempong Baru Kecamatan Upaya pencegahan dan penanggulangan anemia dilakukan dengan memberikan asupan zat besi yang cukup ke dalam tubuh untuk meningkatkan pembentukan hemoglobin. Cara-cara yang dapat dilakukan berupa meningkatkan asupan makanan sumber zat besi, fortifikasi bahan makanan dengan zat besi, dan suplementasi zat besi[16]. Berdasarkan 228 \|LENTERA (Jurnal Pengabdian) LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 masyarakat pada umumnya dalam kegiatan ini pada ibu hamil khususnya. masyarakat pada umumnya dalam kegiatan ini pada ibu hamil khususnya. Agar masyarakat indonesia tetap kuat, sehat dan produktif. Upaya ini tidak hanya mencakup sosialisasi tetapi dengan dilakukan interaksi langsung dapat mempengaruhi pengetahuan ibu tentang pentingnya konsumsi tablet Fe selama proses kehamilan.Sebagian besar pengetahuan seseorang diperoleh melalui penglihatan dan pendengaran. Agar masyarakat indonesia tetap kuat, sehat dan produktif. UCAPAN TERIMAKASIH Ucapan terimakasih kami sampaikan kepada STIKes Yarsi Mataram yang telah memfasilitasi terlaksananya kegiatan pengabdian kepada masyarakat ini dan kepada semua ibu hamil yang sudah meluangkan waktunya pada kegiatan ini. SARAN Setelah kegiatan pengabdian kepada masyarakat ini diharapkan ibu hamil terutama ibu hamil trimester III dapat mulai menerapkan semua materi yang diperoleh tentang penanganan pada anemia ibu hamil. HASIL DAN PEMBAHASAN Kegiatan pelaksanaan pengabdian teridiri atas tiga tahap. Tahap pertama merupakan tahap pretest, dimana tim pngabdian membagikan kuesioner kepada 30 peserta ibu hamil. Gambar 2 Penyampaian Materi kepada ibu Hamil mil. Gambar 1 Kegiatan pretes pengisian kuesioner Gambar 1 Kegiatan pretes pengisian kuesioner Selanjutnya dilakukan kegiatan posttest dengan membagikan kuesioner. Hasil posttest menunjukkan terjadi peningkatan pengetahuan, dengan presentase 66,7% ibu hamil memiliki pengetahuan yang baik dan 33,3% ibu hamil memiliki pengetahuan yang cukup tentang penanganan anemia pada ibu hamil. Pendidikan kesehatan tentang anemia pada ibu hamil terbukti dapat mengurangi kejadian anemia pada kehamilan. Tablet Fe selalu dibagikan setiap posyandu atau setiap kontrol kehamilan ke pelayanan kesehatan tetapi juga menegaskan pentingnya konseling, informasi dan edukasi. Gambar 1 Kegiatan pretes pengisian kuesioner Hasil pretest menunjukkan 24,2% ibu hamil memiliki pemahaman yang baik, 44,2% memiliki pengetahuan yang cukup dan 33,4% ibu hamil memiliki pengetahuan yang kurang tentang penanganan anemia. 229 \|LENTERA (Jurnal Pengabdian) SIMPULAN DAN SARAN Simpulan 1. 1. 1.Ibu hamil memahami pengertian anemia dalam kehamilan, tanda anemia dalam kehamilan, keluhan yang sering dialami ibu l, perubahan fisik dan emosional ibu hamil, pemeriksaan kehamilan, pelayanan kesehatan pada ibu hamil, hal-hal yang harus dihindari oleh ibu selama hamil. Pengetahuan merupakan hasil dari tahu danbiasanya terjadi setelah seseorang melakukan penginderaan terhadap objek tertentu [12]. Pada waktu penginderaan sangat dipengaruhi oleh intensitas perhatian persepsi terhadap objek. Dimana pengetahuan seseorang biasanya diperoleh dari pengalaman yang berasal dari berbagai seumber, misalnya media massa, media poster dan lain sebagainya [13]. Pelaksanaan pemberian pendidikan kesehatan untuk meningkatkan pengetahuan bagi masyarakat dengan resiko yang mungkin bisa terjadi dengan keadaan masyarakat sangat perlu dilakukan, selain sebagai penambahan dan peningkatan pengetahuan juga sebagai tindakan untuk mengurangi resiko terjadinya masalah kesehatan bagi 2. 2. 2. Ibu hamil yang mengikuti penyuluhan mengetahui dan memahami pemahaman anemia pada kehamilan yang ditunjukkan dengan adanya peningkatan pengetahuan dan pemahaman ibu setelah diberi penyuluhan dan diskusi. DAFTAR PUSTAKA (1) Kementerian Perencanaan dan Pembangunan Nasional/ Badan Perencanaan dan Pembangunan Nasional. Rencana Pembangunan Jangka Menengah Nasional (RPJMN) 2015-2019. 2014. (2) SDKI 2012. Survey Demografi dan Kesehatan Indonesia 2012. (3) Profil Kesehatan 2015. Kota NTB (4) Kementrian Kesehatan RI. Hasil Utama Riskesdas 2016 (1) Kementerian Perencanaan dan Pembangunan Nasional/ Badan Perencanaan dan Pembangunan Nasional. Rencana Pembangunan Jangka Menengah Nasional (RPJMN) 2015-2019. 2014. (2) SDKI 2012. Survey Demografi dan Kesehatan Indonesia 2012. (3) Profil Kesehatan 2015. Kota NTB (4) Kementrian Kesehatan RI. Hasil Utama Riskesdas 2016 230 \|LENTERA (Jurnal Pengabdian) LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 LENTERA (Jurnal Pengabdian) ISSN 2774-812X (print); ISSN 2774-9274 (online) Vol. 2 No. 2 Juli Tahun 2022 pp. 226-231 (5) Manuaba, I. B. G. (2012. Ilmu Kebidanan, Penyakit Kandungan dan Keluarga Berencana. Jakarta: EGC, 15, 157. Mochtar, R. (2005). Sinopsis obstetri. edisi dua. Jakarta: EGC. (6) World Healt Organization (WHO). 2015. Maternal Mortality (7) World Health Organization. Guideline: Intermittent iron supplementation in preschool and school-age children. World Heal. Organ. 28 (2012). (8) Kementrian Kesehatan Repuplik Indonesia. 2014. Pedomaan Gizi Seimbang. Jakarta : Kemenkes RI. (9) Balitbang Kemenkes RI. 2018. Riset Kesehatan Dasar; RISKESDAS. Jakarta: Balitbang Kemenkes RI (10) Hariyani, S. (2011). Gizi untuk Kesehatan ibu dan anak. Yogyakarta: Graha Ilmu. Kemenkes, R. I. (2014). (11) Profil Kesehatan Indonesia Tahun 2013. Jakarta: Kementerian Kesehatan (10) Hariyani, S. (2011). Gizi untuk Kesehatan ibu dan anak. Yogyakarta: Graha Ilmu. Kemenkes, R. I. (2014). ( ) y , ( ) gy Ilmu. Kemenkes, R. I. (2014). (11) Profil Kesehatan Indonesia Tahun 2013. Jakarta: Kementerian Kesehatan RI (11) Profil Kesehatan Indonesia Tahun 2013. Jakarta: Kementerian Kesehatan RI (11) Profil Kesehatan Indonesia Tahun 2013. Jakarta: Kementerian Kesehatan RI. (11) Profil Kesehatan Indonesia Tahun 2013. Jakarta: Kementerian Kesehatan RI. ( ) (12) Kristiyanasari, W. (2012). Gizi ibu hamil. Yogyakarta: Nuha Medika. Manuaba, I. (1998). Kapita Selekta Kedokteran. edisi ketiga. Jakarta: EGC. (13) Notoatmodjo, S. (2012). Metodologi penelitian kesehatan. https://jurnalempathy.com/index.php/jurnalempathy/ \| 74 (14) Purwaningtyas, M. L., & Prameswari, G. N. (2017). Faktor kejadian anemia pada ibu hamil. HIGEIA (Journal of Public Health Research and Development), 1(3), 43– 54. (15) Saifuddin, A. B. (2002). Buku panduan praktis pelayanan kesehatan maternal dan neonatal. Yayasan Bina Pustaka Sarwono Prawiroharjo. DAFTAR PUSTAKA (16) Setiawati, S., Rilyani, R., Wandini, R., Wardiyah, A., & Aryanti, L. (2014). FaktorFaktor Yang Mempengaruhi Kejadian Anemia Pada Ibu Hamil Di Wilayah Kerja Puskesmas Sekampung Kabupaten Lampung Timur Tahun 2013. Holistik Jurnal Kesehatan, 8(2). (17) Sulistiyanti, A. (2015). Hubungan tingkat pengetahuan ibu hamil tentang anemia dengan kepatuhan konsumsi tablet Fe di wilayah kerja Puskesmas Masaran I Sragen. Jurnal Maternity, 2(2). (18) Sulistyaningsih, Y. (2017). Penatalaksanaan Pendidikan Kesehatan Diit Anemia Ibu Hamil Dengan Masalah Ketidakseimbangan Nutrisi Kurang Dari Kebutuhan Tubuh. IJMS-Indonesian Journal on Medical Science, 4(1). 231 \|LENTERA (Jurnal Pengabdian) 231 \|LENTERA (Jurnal Pengabdian)
https://openalex.org/W2605434096	http://pure.aber.ac.uk/ws/files/11251302/ncomms14914.pdf	English	null	Past penguin colony responses to explosive volcanism on the Antarctic Peninsula	Nature communications	2,017	cc-by	22,735	Citation for published version (APA): Roberts, S. J., Monien, P., Foster, L. C., Loftfield, J., Schnetger, B., Pearson, E., Juggins, S., Hocking, E., Fretwell, P., Ochyra, R., Haworth, A., Allen, C., Moreton, S., Davies, S., Brumsack, H.-J., Bentley, M. J., Hodgson, D. A., & Ireland, L. (2017). Past penguin colony responses to explosive volcanism on the Antarctic Peninsula. Nature Communications, 8, Article 14914. https://doi.org/10.1038/ncomms14914 Past penguin colony responses to explosive volcanism on the Antarctic P i l Past penguin colony responses to explosive volcanism on the Antarctic Peninsula Roberts, Stephen J.; Monien, Patrick; Foster, Louise C.; Loftfield, Julia; Schnetger, Bernard; Pearson, E.; Juggins, S.; Hocking, Emma; Fretwell, Peter; Ochyra, Ryszard; Haworth, Anna; Allen, Clare; Moreton, Steven; Davies, Sarah; Brumsack, Hans-Jurgen; Bentley, Michael J.; Hodgson, Dominic A.; Ireland, Louise Published in: Nature Communications DOI: 10.1038/ncomms14914 Publication date: 2017 Published in: Nature Communications DOI: 10.1038/ncomms14914 Citation for published version (APA): Roberts, S. J., Monien, P., Foster, L. C., Loftfield, J., Schnetger, B., Pearson, E., Juggins, S., Hocking, E., Fretwell, P., Ochyra, R., Haworth, A., Allen, C., Moreton, S., Davies, S., Brumsack, H.-J., Bentley, M. J., Hodgson, D. A., & Ireland, L. (2017). Past penguin colony responses to explosive volcanism on the Antarctic Peninsula. Nature Communications, 8, Article 14914. https://doi.org/10.1038/ncomms14914 Document License CC BY General rights i h d General rights Copyright and moral rights for the publications made accessible in the Aberystwyth Research Portal (the Institutional Repository) are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain Y f l di t ib t th URL id tif i th bli ti i th Ab t th R h P t l • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of private study or research. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purp research esearch. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal arch. • You may not further distribute the material or use it for any profit-making activity or commercial ga Y f l di t ib t th URL id tif i th bli ti i th Ab t th R h P t l • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal You may not further distribute the material or use it for any profit making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal 1 British Antarctic Survey (BAS), Natural Environmental Research Council (NERC), High Cross, Madingley Road, Cambridge CB3 0ET, UK. 2 Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl-von-Ossietzky-Str. 9–11, 26133 Oldenburg, Germany. 3 Department of Geosciences, Bremen University, Klagenfurter Str. 2–4, 28359 Bremen, Germany. 4 School of Geography, Politics and Sociology, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK. 5 Department of Geography, Northumbria University, Ellison Building, Newcastle-upon-Tyne NE1 8ST, UK. 6 Institute of Botany, Polish Academy of Sciences, ul. Lubicz 46, 31–512 Krako´w, Poland. 7 School of Earth and Ocean Sciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK. 8 NERC Radiocarbon Facility (Environment), Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride, Scotland G75 OQF, UK. 9 Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth SY23 3DB, UK. 10 Department of Geography, Science Laboratories, Durham University, South Road, Durham DH1 3LE, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.J.R. (email: sjro@bas.ac.uk) or to P.M. (email: monien@icbm.de). Take down policy Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. tel: +44 1970 62 2400 email: is@aber.ac.uk tel: +44 1970 62 2400 email: is@aber.ac.uk tel: +44 1970 62 2400 email: is@aber.ac.uk Download date: 24. Oct. 2024 Past penguin colony responses to explosive volcanism on the Antarctic Peninsula Stephen J. Roberts1,, Patrick Monien2,3,, Louise C. Foster1,4,, Julia Loftﬁeld2, Emma P. Hocking5, Bernhard Schnetger2, Emma J. Pearson4, Steve Juggins4, Peter Fretwell1, Louise Ireland1, Ryszard Ochyra6, Anna R. Haworth1,7, Claire S. Allen1, Steven G. Moreton8, Sarah J. Davies9, Hans-Ju¨rgen Brumsack2, Michael J. Bentley10 & Dominic A. Hodgson1,10 Stephen J. Roberts1,, Patrick Monien2,3,, Louise C. Foster1,4,, Julia Loftﬁeld2, Emma P. Hocking5, Bernhard Schnetger2, Emma J. Pearson4, Steve Juggins4, Peter Fretwell1, Louise Ireland1, Ryszard Ochyra6, Anna R. Haworth1,7, Claire S. Allen1, Steven G. Moreton8, Sarah J. Davies9, Hans-Ju¨rgen Brumsack2, Michael J. Bentley10 & Dominic A. Hodgson1,10 Changes in penguin populations on the Antarctic Peninsula have been linked to several environmental factors, but the potentially devastating impact of volcanic activity has not been considered. Here we use detailed biogeochemical analyses to track past penguin colony change over the last 8,500 years on Ardley Island, home to one of the Antarctic Peninsula’s largest breeding populations of gentoo penguins. The ﬁrst sustained penguin colony was established on Ardley Island c. 6,700 years ago, pre-dating sub-fossil evidence of Peninsula- wide occupation by c. 1,000 years. The colony experienced ﬁve population maxima during the Holocene. Overall, we ﬁnd no consistent relationships with local-regional atmospheric and ocean temperatures or sea-ice conditions, although the colony population maximum, c. 4,000–3,000 years ago, corresponds with regionally elevated temperatures. Instead, at least three of the ﬁve phases of penguin colony expansion were abruptly ended by large eruptions from the Deception Island volcano, resulting in near-complete local extinction of the colony, with, on average, 400–800 years required for sustainable recovery. 1 British Antarctic Survey (BAS), Natural Environmental Research Council (NERC), High Cross, Madingley Road, Cambridge CB3 0ET, UK. 2 Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl-von-Ossietzky-Str. 9–11, 26133 Oldenburg, Germany. 3 Department of Geosciences, Bremen University, Klagenfurter Str. 2–4, 28359 Bremen, Germany. 4 School of Geography, Politics and Sociology, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK. 5 Department of Geography, Northumbria University, Ellison Building, Newcastle-upon-Tyne NE1 8ST, UK. 6 Institute of Botany, Polish Academy of Sciences, ul. Lubicz 46, 31–512 Krako´w, Poland. 7 School of Earth and Ocean Sciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK. 8 NERC Radiocarbon Facility (Environment), Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride, Scotland G75 OQF, UK. 9 Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth SY23 3DB, UK. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE Received 16 Nov 2016 \| Accepted 14 Feb 2017 \| Published 11 Apr 2017 Received 16 Nov 2016 \| Accepted 14 Feb 2017 \| Published 11 Apr 2017 ARTICLE ) GC 47 Anvers Trough PD BI James Ross Island HB Weddell Sea Bransfie ld St ra it GC114 GC57 1/2/4/6/7 11/12 Penguin Record type Temperature Sea ice Tephra 0 250 500 km b © DigitalGlobe, Inc., All Rights Reserved 15 10 20 25 30 40 55 45 5 60 50 35 40 25 30 5 10 5 20 40 60 45 20 50 15 35 20 15 35 45 15 10 25 55 45 35 35 20 10 5 60 30 30 10 10 15 30 35 50 30 25 15 15 25 15 25 20 40 5 30 30 20 20 25 30 40 20 25 40 25 30 5 10 5 5 15 5 4 0 30 45 30 62°12'30"S 62°13'S Ardley Island Yanou Lake (YAN) Ardley Lake (ARD) Current penguin nesting area AD3 AD4 58°57'W 58°56'W Raised beaches 13.4–15 m 10.5–11.1 m 4.5–6.6 m d Y5 Y2 Y4 G Sinks Meltwater streams Lakes/ponds Ardley Lake Lake catchments 0 0.5 km 1 14 8 9 5 3 60°S 65°S 65°S 70°S 60°S 3 60°W PC460/1 a South Shetland Islands Deception Island Po la r F r ont Sept. (max.) Sept. Nov. Dec. Oct. Nov. Oct. Dec. F e b . ( m i n . ) GC 47 Anvers Trough PD BI James Ross Island HB Weddell Sea Bransfie ld St ra it GC114 GC57 1/2/4/6/7 11/12 Penguin Record type Temperature Sea ice Tephra 0 250 500 km b © DigitalGlobe, Inc., All Rights Reserved 58°56'W 59°0'W Fil de s P en insula 62°12'S 62°14'S c Maxwell Bay Fild es S t r ait d ARD YAN 0.15–0.4 0.4–1 0.1–0.15 NDVI Index 2 km 0 1 c King George Island Ardley Island b 12 13 © DigitalGlobe, Inc., All Rights Reserved 58°56'W 59°0'W Fil de s P en insula 62°12'S 62°14'S c Maxwell Bay Fild es S t r ait d ARD YAN 0.15–0.4 0.4–1 0.1–0.15 NDVI Index 2 km 0 1 c King George Island Ardley Island b . 12 13 © DigitalGlobe, Inc., All Rights Reserved c King George Island b 12 13 b a Maxwell Bay Figure 1 \| Study sites and the location of different types of records examined. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 explosive (Volcanic Explosivity Index (VEI)25 of 6–7), producing an estimated 30–60 km3 of volcanic ash24,26, but the 20 þ eruptions of the ‘historical’ era (last c. 200 years) and the 30 þ eruptions identiﬁed in Holocene (11.75–0 ka) records have been smaller and less explosive. Most small eruptions from Deception Island present an immediate hazard to penguins nesting within its crater and in the immediate vicinity from ash-fall and pyroclastic ﬂows. However, ﬁne volcanic ash is often widely dispersed across the AP by strong Southern Westerly winds20,22,27,28. For example, following the December 1967–1970 CE eruptions (c. 0.1 km3, VEI ¼ 3)20, a ﬁne layer of tephra o2 mm thick was deposited on Ardley Island and the Fildes Peninsula, King George Island and South Shetland Islands (SSI), c. 120 km north-west of Deception Island (Fig. 1a)21, and in the James Ross Island (JRI) ice core, c. 200 km away on the north-eastern AP29. Even relatively minor E E vidence of different penguin species’ responses to changes in climate and other environmental variables are based on short-term observational records (typically the last 30–60 years1–8), studies of the age and provenance of sub-fossils found in abandoned penguin colonies9–16, and genetic and genomic studies of their evolutionary history17–19. Most of these studies have focussed on how climatic, oceanographic and anthropogenic factors affect penguin populations through access to food and nesting sites and predator–prey dynamics. As far as we are aware, no studies have investigated the long-term impact of large explosive volcanic eruptions on penguin colony size and distribution. Deception Island off the north-western Antarctic Peninsula (AP) (Fig. 1a) is a highly active volcano20–24. ARTICLE Its Late Pleistocene- early Holocene, caldera-forming eruption(s) were exceptionally 15 10 20 25 30 40 55 45 5 60 50 35 40 25 30 5 10 5 20 40 60 45 20 50 15 35 20 15 35 45 15 10 25 55 45 35 35 20 10 5 60 30 30 10 10 15 30 35 50 30 25 15 15 25 15 25 20 40 5 30 30 20 20 25 30 40 20 25 40 25 30 5 10 5 5 15 5 4 0 30 45 30 62°12'30"S 62°13'S Ardley Island Yanou Lake (YAN) Ardley Lake (ARD) Current penguin nesting area AD3 AD4 58°57'W 58°56'W Raised beaches 13.4–15 m 10.5–11.1 m 4.5–6.6 m d Y5 Y2 Y4 G Sinks Meltwater streams Lakes/ponds Ardley Lake Lake catchments 0 0.5 km 58°56'W 59°0'W Fil de s P en insula 62°12'S 62°14'S c Maxwell Bay Fild es S t r ait d ARD YAN 0.15–0.4 0.4–1 0.1–0.15 NDVI Index 2 km 0 1 c King George Island Ardley Island 1 14 8 9 5 3 60°S 65°S 65°S 70°S 60°S 3 60°W PC460/1 a b South Shetland Islands Deception Island Po la r F r ont Sept. (max.) Sept. Nov. Dec. Oct. Nov. Oct. Dec. F e b . ( m i n . ) GC 47 Anvers Trough PD BI James Ross Island HB Weddell Sea Bransfie ld St ra it GC114 GC57 1/2/4/6/7 11/12 12 13 Penguin Record type Temperature Sea ice Tephra 0 250 500 km b © DigitalGlobe, Inc., All Rights Reserved © DigitalGlobe, Inc., All Rights Reserved Figure 1 \| Study sites and the location of different types of records examined. (a) Antarctica, the northern Antarctic Peninsula (AP), the Anvers Trough marine core GC047 sampling site and the South Shetland Islands (SSI), located between the mean modern-day (NSIDC 1981–2010 CE; 20% sea-ice coverage) Austral summer minimum (February or Feb.) and winter maximum (September or Sept.) sea-ice limits (solid white lines) in the Bransﬁeld Strait area. The minimum (Feb.) sea-ice extent on the western side of the AP is located off-image further south of the December (Dec.) sea-ice extent (sea-ice data are from https://nsidc.org/data/seaice_index/). This ﬁgure illustrates how King George Island becomes sea-ice-free before other parts of the SSI and AP, enabling earlier nearshore sea-ice-edge foraging during the Austral spring/summer; locations 1–13 refer to penguin sub-fossil data shown in Fig. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE 5c and listed in Supplementary Data 5; other marine core locations used in tephra analysis are shown in Supplementary Fig. 20; BI ¼ Beak Island, HB ¼ Hope Bay, PD ¼ Palmer Deep; (b) King George Island (KGI) showing the location of records examined in this study, with legend as in a; (c,d) Fildes Peninsula, Ardley Island and the study sampling sites at Yanou Lake (YAN, on KGI), Ardley Lake (ARD) and previous study sites (Y2, Y4, G, AD3, AD4) (refs 42–44) on Ardley Island where bio-geochemical evidence of penguin occupation exists, and the location of meltwater pond Y5. The catchment area of Ardley Lake is c. 66,250 m2 and has an elevation range of c. 16–60 m above present sea level (m a.p.s.l.; mean elevation: 31±10 m a.p.s.l.) deﬁned from the King George Island Digital Elevation Model (KGI-DEM). Published raised beach elevations are in m a.p.s.l. as deﬁned in ref. 85. Vegetated areas in c were mapped from a Normalized Difference Vegetation Index (NDVI) analysis of satellite data acquired on 4 November 2013. See Supplementary Note 3. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. 15 10 20 25 30 40 55 45 5 60 50 35 40 25 30 5 10 5 20 40 60 45 20 50 15 35 20 15 35 45 15 10 25 55 45 35 35 20 10 5 60 30 30 10 10 15 30 35 50 30 25 15 15 25 15 25 20 40 5 30 30 20 20 25 30 40 20 25 40 25 30 5 10 5 5 15 5 4 0 30 45 30 62°12'30"S 62°13'S Ardley Island Yanou Lake (YAN) Ardley Lake (ARD) Current penguin nesting area AD3 AD4 58°57'W 58°56'W Raised beaches 13.4–15 m 10.5–11.1 m 4.5–6.6 m d Y5 Y2 Y4 G Sinks Meltwater streams Lakes/ponds Ardley Lake Lake catchments 0 0.5 km 1 14 8 9 5 3 60°S 65°S 65°S 70°S 60°S 3 60°W PC460/1 a South Shetland Islands Deception Island Po la r F r ont Sept. (max.) Sept. Nov. Dec. Oct. Nov. Oct. Dec. F e b . ( m i n . Past penguin colony responses to explosive volcanism on the Antarctic Peninsula 10 Department of Geography, Science Laboratories, Durham University, South Road, Durham DH1 3LE, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.J.R. (email: sjro@bas.ac.uk) or to P.M. (email: monien@icbm.de). 1 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| w ARTICLE ARTICLE Ardley Island to provide a long-term and continuous record of changes in penguin occupation, because, unlike other shallow ponds on the island (Lake Y1, G, AD3 and AD4, Supplementary Notes 1–3), it has remained above sea-level for the last c. 9 ka (refs 45,46) (Fig. 1d). volcanic eruptions can be potentially devastating to the ecology at locations far from the eruption source30. In terms of penguin mortality, the ﬁne basaltic-andesitic tephra, most commonly produced by Deception Island eruptions, contains large volumes of ﬁne and physically abrasive glass, poisonous aerosols (for example, SO2, F), as well as trace elements and toxic metals (for example, Cd, As, Pb)31 that can adversely affect their physiological functions. ( ) ( g ) Our control site, Yanou Lake (62 13.243 S, 58 57.591 W; c. 11 m a.p.s.l.), located opposite Ardley Lake on Fildes Peninsula, is a former shallow marine basin, submerged below sea level until c. 6.5 ka, with basal sediments deposited in a glaciomarine environment between c. 12 and 7.8 ka (ref. 45). The marine embayment evolved into a shallow near-shore lagoonal basin c. 6.5–6.0 ka, and, when sea-level fell below the retaining sill at c. 14.5.–11 m a.p.s.l. (max.–min. elevation; ref. 45). Yanou Lake was formed and then developed into a predominantly freshwater lake until the present day. As there is no evidence of past or present penguin occupation in the Yanou Lake catchment, this record enabled us to separate the bio-geochemical inputs associated with penguin guano in the Ardley Lake record from those associated with the underlying geology, soils, tephra deposition and natural lake development. Gentoo penguin (Pygoscelis papua) populations across the AP and elsewhere have remained stable or increased over the last 30 years5,32,33, while more ‘sea-ice dependent’ species such as Ade´lie penguins (Pygoscelis adeliae) have declined32,34. These changes have been linked to ‘recent’ regional atmospheric and oceanographic warming on the AP7,8. Conversely, Ade´lie colony populations are increasing in parts of Antarctica (for example, the Ross Sea) where sea-ice is currently expanding35, or have increased during previous colder periods36. Recent AP-wide reductions in land and sea-ice extent/seasonality have altered access routes to breeding sites and have also caused changes in prey availability by shifting the location and distribution of northern-AP shelf-edge krill spawning grounds1,3,8,9,35,37. ARTICLE Since AP krill biomass has not changed signiﬁcantly over the same period35,38, recent changes could potentially relate to the early– mid twentieth century ‘krill-surplus’, which resulted from a reduction in predation pressure on krill due to the exploitation of large numbers of seals and whales in the late nineteenth century to mid twentieth century16,38,39. Gentoo penguins prefer to breed in ice-free areas, and are generalist, inshore predators (foraging within c. 20–30 km of their breeding sites), compared to the more krill-dependent, wider-ranging Ade´lie penguins, while reduced competition from other species on the AP has increased the availability of gentoo prey species across all trophic levels4,16,40. Intriguingly, genetic studies have shown that recent AP species- speciﬁc population trends are different from those of previous warm-periods, but that the response of gentoo penguins to warming is consistently positive16,17. In this study, we undertook detailed geochemical and multi- proxy analyses on an 8,500-year lake sediment record from Ardley Lake (ARD) and compared the results with a lake sediment record from Yanou Lake (YAN). Using variations in inorganic bio-element geochemistry, we established a novel method to determine the proportion of guano present in the ARD and YAN lake sediments—the ornithogenic sediment fraction or Fo.sed.. By converting the percentage Fo.sed. into guano-inﬂuenced sediment dry mass accumulation rates, we modelled estimates of penguin population change for the Ardley Lake colony (see Methods for details). We then determined local- regional drivers of past colony change, by comparing results from the ARD and YAN records with Peninsula-wide records of: (1) lacustrine and sub-fossil evidence of past penguin colony presence11,43; (2) palaeoclimate29, palaeoceanographic47,48 and relative sea level (RSL)45 change, including the ﬁrst AP lake sediment biomarker-based quantitative temperature reconstruction (Yanou Lake), and a new shelf-edge marine sediment record (Anvers Trough) (Fig. 1a; see Methods for details); (3) explosive volcanism from Deception Island, which we conclude had a signiﬁcant impact on colony population at least three times during the Holocene. Ardley Island in the SSI (Fig. 1c,d) contains one of the largest breeding gentoo penguin colonies in the Antarctic41 with c. 5,000 pairs (c. 1.3% of the current global gentoo population), alongside c. 200 breeding pairs of Ade´lie penguins and o50 breeding pairs of chinstrap penguins (P. antarctica) (Supplementary Fig. 1). ARTICLE Between 1950 and 1997 CE, the north-western AP was one of the most rapidly warming regions in the Southern Hemisphere7, and between 1980 and 2005 CE the number of breeding pairs of gentoo penguins on Ardley Island increased by c. þ 310% (minimum–maximum breeding pair count data), whereas between 1980 and 2014, Ade´lie ( 12%) and Chinstrap ( 3.7%) penguin populations have both declined (Supplementary Fig. 1). As each penguin produces c. 84.5 g of guano per day42, every breeding season c. 139 tonnes (dry mass) of penguin guano is discharged onto Ardley Island42, either accumulating in soils and shallow meltwater ponds and lakes (Fig. 1d), or discharging into the sea. Of these, lakes are protected by a permanent water column and can accumulate sediments without signiﬁcant disturbance. Geochemical analyses of guano signatures in lake sediments can thereby provide long-term records of past penguin presence in their catchments43,44 (Supplementary Note 1). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE (a) Antarctica, the northern Antarctic Peninsula (AP), the Anvers Trough marine core GC047 sampling site and the South Shetland Islands (SSI), located between the mean modern-day (NSIDC 1981–2010 CE; 20% sea-ice coverage) Austral summer minimum (February or Feb.) and winter maximum (September or Sept.) sea-ice limits (solid white lines) in the Bransﬁeld Strait area. The minimum (Feb.) sea-ice extent on the western side of the AP is located off-image further south of the December (Dec.) sea-ice extent (sea-ice data are from https://nsidc.org/data/seaice_index/). This ﬁgure illustrates how King George Island becomes sea-ice-free before other parts of the SSI and AP, enabling earlier nearshore sea-ice-edge foraging during the Austral spring/summer; locations 1–13 refer to penguin sub-fossil data shown in Fig. 5c and listed in Supplementary Data 5; other marine core locations used in tephra analysis are shown in Supplementary Fig. 20; BI ¼ Beak Island, HB ¼ Hope Bay, PD ¼ Palmer Deep; (b) King George Island (KGI) showing the location of records examined in this study, with legend as in a; (c,d) Fildes Peninsula, Ardley Island and the study sampling sites at Yanou Lake (YAN, on KGI), Ardley Lake (ARD) and previous study sites (Y2, Y4, G, AD3, AD4) (refs 42–44) on Ardley Island where bio-geochemical evidence of penguin occupation exists, and the location of meltwater pond Y5. The catchment area of Ardley Lake is c. 66,250 m2 and has an elevation range of c. 16–60 m above present sea level (m a.p.s.l.; mean elevation: 31±10 m a.p.s.l.) deﬁned from the King George Island Digital Elevation Model (KGI-DEM). Published raised beach elevations are in m a.p.s.l. as deﬁned in ref. 85. Vegetated areas in c were mapped from a Normalized Difference Vegetation Index (NDVI) analysis of satellite data acquired on 4 November 2013. See Supplementary Note 3. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Results G Guano geochemistry. The geochemistry of Ardley Lake (ARD, study site) and Yanou Lake (YAN, control site) sediments showed signiﬁcant ﬂuctuations with sediment depth and modelled age (Figs 2–4, Supplementary Notes 4–7, Supplementary Figs 3–13, 16, Supplementary Tables 1–7, Supplementary Data 1). R-mode cluster analysis performed on the inter-correlation coefﬁcients of major and trace element concentrations in the ARD record clearly separated guano-derived elements (Sr, Cu, Zn, Se, Ca, Se, P, C, N, S) and guano-associated elements (Cd, As, Hg) from lithogenic elements (for example, Al, Mg, Si, Sc, Ti, Zr, Y and REE) derived from weathering of local volcanic bedrock (Fig. 2, Supplementary Fig. 8b, Supplementary Table 4). No similarly clear separation of elements was evident in the YAN control record (Supplementary Fig. 8c; Supplementary Table 5). The positive correlation between Ca and P (r ¼ 0.63, Po0.001; Supplementary Table 4) suggests that hydroxylapatite is the main phosphate phase in the ARD record (Supplementary Notes 1,8). Selected element-aluminium cross-plots (Fig. 3a) show that Ardley Lake sediments clearly represent a mixture between two end members: eroded Ardley Island bedrock and ornithogenically derived (bird-formed) soil and sediment from the lake’s catch- ment, whereas the geochemistry of Yanou Lake sediments is Ardley Lake (62 12.774 S, 58 56.398 W; c. 16 metres above present mean sea level, m a.p.s.l., as deﬁned in ref. 45) is the only large (c. 7,270 m2), permanent closed-basin water body on Ardley Island (c. 268,510 m2), and its only permanent depositional ‘sink’ (Fig. 1d, Supplementary Fig. 2). With two prominent meltwater inﬂows, and an overspill outﬂow bounded by its retaining sill at c. 18–16 m a.p.s.l. (max.-min. elevation45), the Ardley Lake catchment area (c. Results G 66,250 m2) is well-situated in the centre of 3 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 0 4 8 Visible tephra deposits Airfall tephra Redeposited tephra Munsell colour 2.5Y 2.5/1 Black 2.5Y 3/3 Dark olive brown 2.5Y 3/1 Very dark grey 2.5Y 3/1 Dark grey 2.5Y 3/2 Very dark greyish brown 2.5Y 4/3 Olive brown 2.5Y 4/2 Dark greyish brown 2.5Y 4/4 Olive brown Sedimentology Silt Clay Sand Tephra-rich (<50% tephra) Tephra bed (>50% tephra) Nodules / peloids Matrix-supported diamicton Clast-supported diamicton Moss bed (>50% moss) Moss fragments (<50% moss) 1 2 4a 5 Penguin bones (B1–B6) and bone fragments 1 3 0.1 0.3 0.5 0.1 0.25 0.4 0 0.4 0.8 Sum of squares CONISS XRF Zones (13) Guano phases 50 150 1 3 5 50 150 M7 M8A M7A Munsell colour Visible tephra Sedimentology Munsell colour Visible tephra Sedimentology Clay Silt Sand Grain size Fine Med. Coarse 0 50 100 150 200 250 300 350 Depth (cm) 0 50 100 150 200 250 300 350 Depth (cm) TC/Al TN/Al TS/Al P/Al Zn/Al (x 10–4) As/Al (x 10–4) Cu/Al (x 10–4) Guano and guano-related elements 4b 3 Diatom zones Lake environment T4 T1 T3 B1 B2 B3/4 B5 B6 B1 B2 B3/4 B5 B6 b c ab T6 T7 T8? T5b T5a T4 T1 T3b c ab T6 T7 T8? T5b T5a Fresh– brackish D1 D2 D3 D4 D5 D6 Fresh– brackish to brackish F.-Brack. Brackish– fresh Fresh– brackish and fresh Fresh and fresh– brackish B6 B1 B2 B3/4 2.6 3.0 0.08 0.12 100 200 300 0.5 1.5 0 0.01 0.02 0 4 8 10 20 30 Depth (cm) Guano and guano-related elements Lithogenic elements Si/Al Ti/Al Sr/Al (x 10–4) Ca/Al Hg/Al (x 10–4) Se/Al (x 10–4) Zr/Al (x 10–4) 0 50 100 150 200 250 300 350 1 3 5 4b 4a 2 Moss Bulk organic (fresh) Lacustrine algae SH13-Terrestrial Guano-influenced sed. MARINE13 (ΔR=700±50) Bulk organic (brackish) Penguin bone–collagen Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 M7 M8A M7A B1 B2 B2.1 B2.2 B4.1 B6.1 B6.2 B6.3 B6.4 B3 B4 B5 B6 Figure 2 \| Whole core geochemistry of Ardley Lake sediments. Downcore proﬁles of selected aluminium normalized guano-elements (P, Zn, As, Cu, TN, TS, TC, Ca, Sr, Hg, Se), lithogenic elements (Si, Ti, Zr) and age-depth model (ARD-M5) of the ARD record (Supplementary Data 1,2). Results G MARINE13 (ΔR=700±50) Bulk organic (brackish) Penguin bone–collagen Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 M7 M8A M7A B1 B2 B2.1 B2.2 B4.1 B6.1 B6.2 B6.3 B6.4 B3 B4 B5 B6 Figure 2 \| Whole core geochemistry of Ardley Lake sediments. Downcore proﬁles of selected aluminium normalized guano-elements (P, Zn, As, Cu, TN, TS TC Ca Sr Hg Se) lithogenic elements (Si Ti Zr) and age depth model (ARD M5) of the ARD record (Supplementary Data 1 2) Intact juvenile Depth (cm) Figure 2 \| Whole core geochemistry of Ardley Lake sediments. Downcore proﬁles of selected aluminium normalized guano-elements (P, Zn, As, Cu, TN, TS, TC, Ca, Sr, Hg, Se), lithogenic elements (Si, Ti, Zr) and age-depth model (ARD-M5) of the ARD record (Supplementary Data 1,2). Intact juvenile Pygoscelis Papua bones found at core depths are labelled B1–B6 (scale bars are 1 cm): B1 is a cnemial crest bone of the tibiotarsus (left leg); at B2, two base occipital condyle bones from a poorly fused skull(s); B3 is a heavily weathered metatarsal (foot) bone; at B4, two weathered carpometacarpus (wing) bones; B5 is a talon; B6 are mid-upper neck cervical vertebrae bones, found intact vertically (4 of 5 bones shown). Bone-collagen was extracted and radiocarbon dated from bones B2.1, B4.1, B6.2 (see Supplementary Fig. 4 and Supplementary Table 2 for details). strongly associated with local bedrock and tephra deposition. The inﬂuence of penguin guano on the sediment samples from Ardley Lake is further supported by their C/N and C/P ratios (C/N ¼ av. 6.6, C/P ¼ av. 3.3), which are close to those previously reported in ornithogenic soils (C/N ¼ 6.3, C/P ¼ 1.2–3.7) (ref. 49) (Fig. 3b). In contrast, they differ signiﬁcantly from ratios found in phyto- plankton and local plants, for example, carpet mosses, lichens (Usnea antarctica), liverworts and vascular plants, such as Deschampsia antarctica (Fig. 3b, C/N ¼ 21–114)50 present in the catchment area of both lakes. Guano phases. Sustained high concentrations of bio-elements (weighted mean Fo.sed.410%) and stratigraphically constrained incremental sum of squares (CONISS) cluster analysis were used to deﬁne ﬁve phases of elevated guano ﬂux (GP-1 to GP-5) (shown as 1–5 in circles in Figs 2, 4 and 5; see Methods for details). Periods of exceptionally elevated guano within each guano phase (darker green shading in Fig. Results G Intact juvenile Pygoscelis Papua bones found at core depths are labelled B1–B6 (scale bars are 1 cm): B1 is a cnemial crest bone of the tibiotarsus (left leg); at B2, two base occipital condyle bones from a poorly fused skull(s); B3 is a heavily weathered metatarsal (foot) bone; at B4, two weathered carpometacarpus (wing) bones; B5 is a talon; B6 are mid-upper neck cervical vertebrae bones, found intact vertically (4 of 5 bones shown). Bone-collagen was extracted and radiocarbon dated from bones B2.1, B4.1, B6.2 (see Supplementary Fig. 4 and Supplementary Table 2 for details). 0 4 8 Visible tephra deposits Airfall tephra Redeposited tephra Munsell colour 2.5Y 2.5/1 Black 2.5Y 3/3 Dark olive brown 2.5Y 3/1 Very dark grey 2.5Y 3/1 Dark grey 2.5Y 3/2 Very dark greyish brown 2.5Y 4/3 Olive brown 2.5Y 4/2 Dark greyish brown 2.5Y 4/4 Olive brown Sedimentology Silt Clay Sand Tephra-rich (<50% tephra) Tephra bed (>50% tephra) Nodules / peloids Matrix-supported diamicton Clast-supported diamicton Moss bed (>50% moss) Moss fragments (<50% moss) 1 2 4a 5 Penguin bones (B1–B6) and bone fragments 1 3 0.1 0.3 0.5 0.1 0.25 0.4 0 0.4 0.8 Sum of squares CONISS XRF Zones (13) Guano phases 50 150 1 3 5 50 150 M7 M8A M7A Munsell colour Visible tephra Sedimentology Munsell colour Visible tephra Sedimentology Clay Silt Sand Grain size Fine Med. Coarse 0 50 100 150 200 250 300 350 Depth (cm) 0 50 100 150 200 250 300 350 Depth (cm) TC/Al TN/Al TS/Al P/Al Zn/Al (x 10–4) As/Al (x 10–4) Cu/Al (x 10–4) Guano and guano-related elements 4b 3 Diatom zones Lake environment T4 T1 T3 B1 B2 B3/4 B5 B6 B1 B2 B3/4 B5 B6 b c ab T6 T7 T8? T5b T5a T4 T1 T3b c ab T6 T7 T8? T5b T5a Fresh– brackish D1 D2 D3 D4 D5 D6 Fresh– brackish to brackish F.-Brack. Brackish– fresh Fresh– brackish and fresh Fresh and fresh– brackish B6 B1 B2 B3/4 2.6 3.0 0.08 0.12 100 200 300 0.5 1.5 0 0.01 0.02 0 4 8 10 20 30 Depth (cm) Guano and guano-related elements Lithogenic elements Si/Al Ti/Al Sr/Al (x 10–4) Ca/Al Hg/Al (x 10–4) Se/Al (x 10–4) Zr/Al (x 10–4) 0 50 100 150 200 250 300 350 1 3 5 4b 4a 2 Moss Bulk organic (fresh) Lacustrine algae SH13-Terrestrial Guano-influenced sed. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Results G 4b) were deﬁned as 4 the upper bound (95% conﬁdence level) of the whole-record weighted mean Fo.sed. value of 27.93% (green dotted line in Fig. 4b). g g The ﬁrst occupation of the Ardley Lake catchment by penguins occurred between 6.7 and 6.3 cal ka BP (GP-1), with further Fo.sed. maxima between 5.8–5.3 (GP-2), 4.5–4.3 (GP-3), 4.0–3.0 (GP-4) and 2.7–1.3 (GP-5) cal ka BP (Figs 4b and 5, Table 1). No similar patterns were found in the control site YAN (Fig. 4a, Supplementary Figs 10,11c). Intact juvenile gentoo penguin bones and bone fragments were found in guano phases GP-2 and GP-4 and in sediments deposited between GP-2 and GP-3 (Fig. 2, Supplementary Fig. 4). Guano dry mass accumulation rates (Fo.sed. DMAR in g cm 2 a 1, thick black line in Fig. 4b) account for variations in the sedimentation rate that reﬂect changes in erosional input into Ardley Lake, and, therefore, do not always correspond to higher guano-inﬂuenced sediment Fo.sed. percentages. Since Fo.sed. DMAR values underpin In the ARD record, in-phase changes in several aluminium- normalized bio (associated) elements, such as P, Zn, As, Cu, Ca, Sr, Hg, and Se, together with TN, TC (equivalent to TOC) and TS were recorded (Fig. 2, Table 1, Supplementary Figs 6–8,11b,16). This enabled us to derive the mean relative proportion of guano or ornithogenically derived sediment (% Fo.sed.) in the Ardley Lake sediment matrix using a mixing equation (equation (1, Methods) based on the element/aluminium (Al) ratio of selected bio-elements (Cu, P, Sr, Zn) in the Ardley Lake sediments, the mean Al-normalized Ardley Island bedrock51 composition and Al-normalized mean composition of guano-bearing ornithogenic sediment and soils49 from King George Island as end members (see Methods for details). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 4 4 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 \| 0 1 2 3 2 4 6 8 10 Al (mass %) a Mg (mass %) 0 0.2 0.4 0.6 0.8 2 4 6 8 10 Al (mass %) Cu (mg g–1) 0 0.2 0.4 0.6 0.8 1.0 2 4 6 8 10 Al (mass %) Zn (mg g–1) 0 5 10 15 20 2 4 6 8 10 Al (mass %) Ca (mass %) 0.5 1.0 1.5 2.0 2.5 2 4 6 8 10 Al (mass %) Sr (mg g–1) 0 2 4 6 8 10 2 4 6 8 10 Al (mass %) P (mass %) r = 0.89 P<0.0001 r = –0.63 P<0.0001 r = –0.90 P<0.0001 r = –0.94 P<0.0001 r = –0.88 P<0.0001 r = –0.90 P<0.0001 0 1 2 3 2 4 6 8 10 Al (mass %) a Mg (mass %) r = 0.89 P<0.0001 0 0.2 0.4 0.6 0.8 2 4 6 8 10 Al (mass %) Cu (mg g–1) r = –0.88 P<0.0001 0 0.2 0.4 0.6 0.8 1.0 2 4 6 8 10 Al (mass %) Zn (mg g–1) r = –0.90 P<0.0001 a Al (mass %) 0 5 10 15 20 2 4 6 8 10 Al (mass %) Ca (mass %) r = –0.63 P<0.0001 0.5 1.0 1.5 2.0 2.5 2 4 6 8 10 Al (mass %) Sr (mg g–1) r = –0.90 P<0.0001 0 2 4 6 8 10 2 4 6 8 10 Al (mass %) P (mass %) r = –0.94 P<0.0001 P (mass %) Ardley Island bedrock Fildes Peninsula bedrock Ardley Lake (ARD) sediments Yanou Lake (YAN) sediments ARD samples Liverworts Moss Lichens Ornithogenic soils (guano) Ornithogenic soils (humus) Ornithogenic soils (phosphatic clay) Vascular plants 0 10 20 30 40 50 0 TC (mass %) 0 10 20 30 40 50 0 1 2 3 4 5 TN (mass %) TC (mass %) r = 0.98 P<0.0001 b Yanou Lake (YAN) sediments Fildes Peninsula bedrock gure 3 \| Scatterplots of element/aluminium ratios and comparison of C/N and C/P ratios. (a) rdley Lake and Yanou Lake sediments (this study), ornithogenic soils49 and local bedrock86. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 ARTICLE ation models, guano phases GP-2, GP-3 and GP-4 corres- to a higher number of penguins present in the Ardley Lake h i Fi b ( M h d S l N details). A good correspondence between the Ardley Lake fossil record and the sub-fossil record of former penguin c f h AP i d i h h d il ARD samples Liverworts Moss Lichens Ornithogenic soils (guano) Ornithogenic soils (humus) Ornithogenic soils (phosphatic clay) Vascular plants 0 10 20 30 40 50 0 2 4 6 8 P (mass %) TC (mass %) 0 10 20 30 40 50 0 1 2 3 4 5 TN (mass %) TC (mass %) r = 0.98 ARD data: linear regression summary statistics Redfield ratio P<0.0001 r = 0.89 P<0.0001 Ardley Lake (ARD) sediments Ornithogenic soils Ardley Island bedrock 0 1 2 3 2 4 6 8 10 Al (mass %) a b Mg (mass %) 0 0.2 0.4 0.6 0.8 2 4 6 8 10 Al (mass %) Cu (mg g–1) 0 0.2 0.4 0.6 0.8 1.0 2 4 6 8 10 Al (mass %) Zn (mg g–1) 0 5 10 15 20 2 4 6 8 10 Al (mass %) Ca (mass %) 0.5 1.0 1.5 2.0 2.5 2 4 6 8 10 Al (mass %) Sr (mg g–1) 0 2 4 6 8 10 2 4 6 8 10 Al (mass %) P (mass %) Yanou Lake (YAN) sediments Fildes Peninsula bedrock r = 0.89 P<0.0001 r = –0.63 P<0.0001 r = –0.90 P<0.0001 r = –0.94 P<0.0001 r = –0.88 P<0.0001 r = –0.90 P<0.0001 ARD: linear regression summary statistics 3 \| Scatterplots of element/aluminium ratios and comparison of C/N and C/P ratios. (a) Scatterplots of selected element/aluminium Lake and Yanou Lake sediments (this study), ornithogenic soils49 and local bedrock86. (b) Total Carbon/Total Nitrogen (TC/TN) and To n/Phosphorus (TC/P) ratios of Ardley Lake sediments, local lichens (for example, Usnea antarctica)50, liverworts (Cephalozia varians), mos ar plants (Deschampsia antarctica)50 and different types of ornithogenic soils49. The solid and the dashed lines show linear regression lines of ore ARD, and C/N and C/P ratios of phytoplankton according to the Redﬁeld ratio, respectively (Supplementary Data 3). ARTICLE and JRI atmospheric temperature (Fig. 4f)29. Overall, none of the guano phases were statistically related to warmer sea surface temperatures in the Palmer Deep (PD) record. Conversely, the non-guano phases were associated with signiﬁcantly warmer sea surface temperatures in the Palmer Deep record (PD-SST 0–200 m; Fig. 4e)48 (two-tailed Mann–Whitney U-tests: YAN-GDGT: P ¼ 0.58, JRI: P ¼ 0.57, PD-SST: P ¼ 0.01; Supplementary Discussion, Supplementary Palaeoenvironmental records. We then compared these ﬁve phases of elevated guano ﬂux with a range of local and regional records of past environments to identify the major drivers of changes in penguin populations. First, we found no signiﬁcant difference (5 or 10% level) in temperature data during guano and non-guano phases from cross-northern Peninsula 9–0 ka terrestrial temperature records, YAN-GDGT reconstructed mean summer air temperatures (MSAT) (Fig. 4a) Warm SST (0–200 m) Warm surface SST SST (°C) 0 2 Palmer Deep Temp. Record +Warm +Warm 0–200 m c Maxwell Bay Sea-ice Record TOC (%) 0.0 0.6 0.3 +Open water d Diatom ratio (F. curta/F. kerg.) Pelagic open water diatoms (%) 0 10 20 30 Anvers Shelf Sea-ice Record 0 1 2 3 1 2 4 5 3 Yanou Lake Temp. Record a GDGT-MSAT 6–0 ka Anomaly (°C ±1σ) +Warm (MSAT) Ardley Lake b e +Sea ice +Open water –1 0 1 PCA2μ-XRF (9%) T8? 0.00 0.05 0.10 ? T3b T1 0 10 20 SandDMAR (g cm–2 a–1) Fo.sed. DMAR (g cm–2 a–1) SandDMAR (g cm–2 a–1) T2 T4 T5 T6 T3a 0 0.05 Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 g AP Warm periods RRR MCA? f James Ross Island Ice Core Temp. Record JRI temp. anomaly (°C ±1σ) –1 0 1 Airfall/ tephra and reworked sediment T7 6–0 ka upper bound on mean (95%) 100-year smoothed <10 °C 6–0 ka weighted mean MHH EHO to 11.5 ka 10% 0% % Guano (Fo.sed. % ±1σ) 95% conf. upper bound 0 50 100 Guano phases Guano phases 1 4 5 3 2 Volcanic eruptions 0.00 0.01 0.02 0.03 %Fo.sed. 100-year Fo.sed. ARTICLE DMAR –5 0 5 10 +Brackish conditions / >wind Glaciomarine to 11 ka Palaeoenvironmental data >6–0 ka upper bound on mean (95%): Open water 6-0 ka weighted mean 6–0 ka upper bound on mean (95%) 6–0 ka lower bound on mean (95%) >6–0 ka upper bound on mean (95%): Discontinuou >6–0 ka upper bound on mean (95%): Sustained w >2x 6–0 ka upper bound on mean (95%): Sustained AP composite warm periods Volcanic eruptions VEI>4 VEI>3 VEI< or =3 Weighted mean Eruption age and 95% min.–max. (cal a BP) Visible tephra No guano Ardley Lake post-eruption colony recovery interval ARD Guano phases GP-1 to GP-5 Penguin data ARD exceptionally elevated guano phases Published bone and egg ages (cal a ± 1σ) Diatom zone boundaries Modelled ARD bone ages (cal a ± 95% min.–max.) ARD bone–collagen ages (cal a ± 1σ) Marine 100% Marine 100% Lacustrine Brackish Brackish Colder C lder o e d o e Colder er Warme glaciating Deg RSL<5 m 5 m Penguin Record NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10 1038/ncomms14914 \| www nature com/naturecom Yanou Lake Temp. Record a GDGT-MSAT 6–0 ka Anomaly (°C ±1σ) +Warm (MSAT) Ardley Lake b –1 0 1 PCA2μ-XRF (9%) T8? 0.00 0.05 0.10 ? T3b T1 0 10 20 SandDMAR (g cm–2 a–1) Fo.sed. DMAR (g cm–2 a–1) SandDMAR (g cm–2 a–1) T2 T4 T5 T6 T3a 0 0.05 Airfall/ tephra and reworked sediment T7 6–0 ka upper bound on mean (95%) 100-year smoothed <10 °C 6–0 ka weighted mean 10% 0% % Guano (Fo.sed. % ±1σ) 95% conf. upper bound 0 50 100 Guano phases 1 4 5 3 2 Volcanic eruptions 0.00 0.01 0.02 0.03 %Fo.sed. 100-year Fo.sed. DMAR –5 0 5 10 Glaciomarine to 11 ka Marine 100% Marine 100% Lacustrine Brackish Brackish Colder C lder o e d o e Colder er Warme glaciating Deg RSL<5 m 5 m Penguin Record Yanou Lake Temp. Record a Warm SST (0–200 m) Warm surface SST SST (°C) 0 2 Palmer Deep Temp. Record +Warm +Warm 0–200 m c Maxwell Bay Sea-ice Record TOC (%) 0.0 0.6 0.3 +Open water d Diatom ratio (F. curta/F. kerg.) Pelagic open water diatoms (%) 0 10 20 30 Anvers Shelf Sea-ice Record 0 1 2 3 1 2 4 5 3 e +Sea ice +Open water Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 g AP Warm periods RRR MCA? NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 (b) T arbon/Phosphorus (TC/P) ratios of Ardley Lake sediments, local lichens (for example, Usnea anta ARD samples Liverworts Moss Lichens Ornithogenic soils (g Ornithogenic soils (h Ornithogenic soils (p Vascular plants 0 10 20 30 40 50 0 1 2 3 4 5 TN (mass %) TC (mass %) r = 0.98 P<0.0001 b Yanou Fildes Peninsula bedrock \| Scatterplots of element/aluminium ratios and comparison of C ake and Yanou Lake sediments (this study) ornithogenic soils49 a b 0 10 20 30 40 50 0 2 4 6 8 P (mass %) TC (mass %) r = 0.89 P<0.0001 Redfield ratio Figure 3 \| Scatterplots of element/aluminium ratios and comparison of C/N and C/P ratios. (a) Scatterplots of selected element/aluminium ratios of Ardley Lake and Yanou Lake sediments (this study), ornithogenic soils49 and local bedrock86. (b) Total Carbon/Total Nitrogen (TC/TN) and Total Carbon/Phosphorus (TC/P) ratios of Ardley Lake sediments, local lichens (for example, Usnea antarctica)50, liverworts (Cephalozia varians), mosses50, vascular plants (Deschampsia antarctica)50 and different types of ornithogenic soils49. The solid and the dashed lines show linear regression lines of samples from core ARD, and C/N and C/P ratios of phytoplankton according to the Redﬁeld ratio, respectively (Supplementary Data 3). population models, guano phases GP-2, GP-3 and GP-4 corres- pond to a higher number of penguins present in the Ardley Lake catchment shown in Fig. 5b (see Methods, Supplementary Note 8, Supplementary Figs 16–19, Supplementary Table 9 for more details). A good correspondence between the Ardley Lake guano/ fossil record and the sub-fossil record of former penguin colonies from across the AP exists during these phases, and until the end of guano phase GP-5, c. 1.3 cal ka BP (Fig. 5). 5 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunication NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 ARTICLE % ¼ 43.66±15.37 (1s) %), occurred between 3.4 and 3.0 cal ka BP when both the YAN-GDGT and JRI records were signiﬁcantly warmer than their 6–0 ka means (Fig. 4, Supplementary Fig. 18, Supplementary Tables 7,8). Second, we compared the guano phases with sea-ice and open water proxies, which impact on the penguins’ access to nesting sites and prey species. Although guano-phase GP-4 coincided with a sustained period of reduced spring/summer open water seasonality in the Anvers Trough record from c. 4 cal ka BP onwards (Fig. 4d), there were no signiﬁcant differences in open water conditions in Maxwell Bay (P ¼ 0.26) or at the Anvers Shelf (P ¼ 0.25) for guano and non-guano phases. More prolonged spring/summer open water conditions existed at the Anvers Trough shelf-edge in mid-Holocene guano phases (GP-2 and GP-3), but not during late Holocene guano phases (GP-4 and GP-5) (P ¼ 0.10). Volcanic activity. Third, we compared the guano phases with the combined record of volcanic tephra in the ARD and YAN lake sediment records. We identiﬁed seven phases of VEI ¼ 3 or VEI43 eruption activity (T1–T7) (Fig. 4a,b). Eruption T7 aside, Two other VEI ¼ 43 eruptions (T3a, T2) occurred at the same time as measurable, but not-signiﬁcant reductions in the Table 1 \| Summary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment percentage data. ARD ARD ARD Calibrated ages (cal a BP) Duration n % Guano-inﬂuenced sediment CONISS zone Guano phase Core depth min.–max. (cm) Weighted mean age min.–max. 95% conf. age range min.–max. (years±95% conf. diff.) [95% min.–max.] Weighted mean Fo.sed. (%±1r) 95% Conf. Fo.sed. Interval (lower–upper %) 12 5 13–35 1,290–2,670 1,160–2,830 1,380±145 [1,670] 11 23.57±10.74 16.35–30.79 9 4b 65–112 3,000–3,430 2,880–3,580 1,010±170 [1,350] 26 43.66±15.37 39.47–47.85 10 4a 112–155 3,430–4,010 3,270–4,230 26 12.95±2.76 11.41–14.49 7 3 175–190 4,310–4,510 4,090–4,640 200±175 [550] 7 26.33±12.16 18.92–33.74 5 2 284–310 5,300–5,750 5,180–6,020 450±195 [840] 9 29.38±9.90 21.95–36.82 3 1 320–326 6,270–6,670 5,820–7,360 400±570 [1,540] 3 26.77±9.49 5.31–48.23 Summary Whole record — 198 25.31±9.41 22.69–27.93 statistics Guano phases 690±250 [1,190] 82 34.19±12.19 31.06–37.32 Non-guano phases — 116 3.56±2.49 2.42–4.69 Late Holocene guano phases 1,200±160 [1,510] 62 36.57±12.73 32.73–40.40 Mid Holocene guano phases 350±310 [980] 20 27.77±10.72 23.66–31.88 All ages are in calibrated (equivalent to calendar) years before present (cal a BP), where BP is deﬁned as 1950 CE (see Supplementary Methods); [95% min. ARTICLE (cal years) is the average of the weighted mean minimum 95% conﬁdence minimum ages and the 95% conﬁdence maximum – weighted mean maximum ages; the duration of the 95% conﬁdence minimum to maximum age range is shown in square brackets. Guano-inﬂuenced sediment (Fo.sed.) weighted mean percentages ±1s, signiﬁcance level a ¼ 0.05.. Figure 4 \| Penguin occupation phases in the Ardley Lake record compared with key records of volcanic activity climate and sea-ice from the Antarctic Peninsula region for the last 10,000 years. In order from top: (a) Yanou Lake (YAN) sediment core palaeoenvironmental summary, reconstructed mean summer air temperature (MSAT) GDGT 100-year temperature anomaly data; 200 mm micro-XRF (m-XRF) Principal Component Analysis 2nd axis (PCA2, 9% variance explained) and sand Dry Mass Accumulation Rate (DMAR); main visible tephra deposits are black diagonally hatched zones, dotted lines show correlation of tephra layers between the ARD and YAN records, circle size reﬂects Volcanic Explosivity Index (VEI) of major eruptions and circle position is the weighted mean modelled age with 95% min.–max. conﬁdence age ranges (grey horizontal bars) based on 2.5% and 97.5% quantiles from Bayesian age-depth modeling (see Supplementary Notes 5–7 and Supplementary Methods). (b) Numbered, green-shaded guano phases GP-1 to GP-5 with the calculated fractions of ornithogenic sediments in the Ardley Lake record (Fo.sed. %±1s errors in grey) overlain by Fo.sed. dry mass accumulation rate (DMAR) (thick black line, 1s errors not shown for clarity, but in GP-4 and GP-5 these are on average c. 25-35%); red stippled areas are recovery intervals; white squares are radiocarbon-dated penguin bone-collagen extract ages; grey squares are modelled ages of bones in the ARD record. (c) Maxwell Bay (MB) percentage Total Organic Carbon (TOC) data (in grey) and 100-year interval negative exponential smoothed data (dark blue) used as sea-ice proxy52; data 46–0 ka upper bound on the mean (95% conﬁdence level) represents increased open water conditions (shaded dark blue). (d) Anvers Trough GC047/TC046 sediment core: the commonly used diatom-based ratio (Fragilariopsis curta: Fragilariopsis kerguelensis) with data 6–0 ka upper bound on the mean (95% conﬁdence level) shaded dark blue representing increased open-water conditions and percentage of total spring/summer pelagic open- water diatoms species (red line). ARTICLE f James Ross Island Ice Core Temp. Record JRI temp. anomaly (°C ±1σ) –1 0 1 MHH EHO to 11.5 ka Guano phases +Brackish conditions / >wind Palaeoenvironmental data >6–0 ka upper bound on mean (95%): Open water 6-0 ka weighted mean 6–0 ka upper bound on mean (95%) 6–0 ka lower bound on mean (95%) >6–0 ka upper bound on mean (95%): Discontinuous warmth >6–0 ka upper bound on mean (95%): Sustained warmth >2x 6–0 ka upper bound on mean (95%): Sustained warmth AP composite warm periods Volcanic eruptions VEI>4 VEI>3 VEI< or =3 Weighted mean Eruption age and 95% min.–max. (cal a BP) Visible tephra No guano Ardley Lake post-eruption colony recovery interval ARD Guano phases GP-1 to GP-5 Penguin data ARD exceptionally elevated guano phases Published bone and egg ages (cal a ± 1σ) Diatom zone boundaries Modelled ARD bone ages (cal a ± 95% min.–max.) ARD bone–collagen ages (cal a ± 1σ) \| \| \| NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 mid-late Holocene tephra layers had well-deﬁned basaltic- andesitic compositions, typical of Deception Island source com- positions (Supplementary Figs 20,21, Supplementary Table 10). Three VEI43 eruption events (T5a, T4, T3b) coincided with immediate and signiﬁcant reductions in guano deposition (that is, Fo.sed. to o10%) (Fig. 4a,b, Table 1). The most disruptive series of eruptions (T5a,b) recorded in both ARD and YAN sediments occurred during the mid-Holocene between c. 5.5–4.9 cal ka BP (combined ARD–YAN 95% maximum to mean age range; Fig. 4, Table 2, Supplementary Figs 12,16, Supplementary Table 3). Brackish conditions, greater catchment destabilization and increased erosion occurred for c. 1,000 years during the T5 eruption series (Fig. 4, Supplementary Fig. 16). The T4 (c. 4.5–4.2 cal ka BP) and T3b (c. 3.2–3.0 cal ka BP) eruptions that followed the T5 eruption series were smaller, but also coincided with immediate and signiﬁcant reductions in guano deposition. Colony recovery after the T5a eruption took 780±50 years [530– 1,130 years], and 410±30 [0–760] years and 480±60 [160–390] years after the T4 and T3b eruptions, respectively, with an overall weighted mean (±s.e.) recovery time of 570±110 years (Table 2). Table 8). Notably, the late Holocene guano Fo.sed. DMAR maxima in guano phase GP-4 (Fo.sed. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE max.] is the minimum and maximum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling (see Supplementary Notes 5–7 and Supplementary Methods); durations are in calendar years with guano phase 4 shown as the combined duration of 4a and 4b; 95% conf. diff. (cal years) is the average of the weighted mean minimum 95% conﬁdence minimum ages and the 95% conﬁdence maximum – weighted mean maximum ages; the duration of the 95% conﬁdence minimum to maximum age range is shown in square brackets. Guano-inﬂuenced sediment (Fo.sed.) weighted mean percentages ±1s, signiﬁcance level a ¼ 0.05.. Table 1 \| Summary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment percentage data. Table 1 \| Summary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment percentage data. mmary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment per All ages are in calibrated (equivalent to calendar) years before present (cal a BP), where BP is deﬁned as 1950 CE (see Supplementary Methods); [95% min. max.] is the minimum and maximum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling (see Supplementary Notes 5–7 and Supplementary Methods); durations are in calendar years with guano phase 4 shown as the combined duration of 4a and 4b; 95% conf. diff. (cal years) is the average of the weighted mean minimum 95% conﬁdence minimum ages and the 95% conﬁdence maximum – weighted mean maximum ages; the duration of the 95% conﬁdence minimum to maximum age range is shown in square brackets. Guano-inﬂuenced sediment (Fo.sed.) weighted mean percentages ±1s, signiﬁcance level a ¼ 0 05 All ages are in calibrated (equivalent to calendar) years before present (cal a BP), where BP is deﬁned as 1950 CE (see Supplementary Methods); [95% min. max.] is the minimum and maximum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling (see Supplementary Notes 5–7 and Supplementary Methods); durations are in calendar years with guano phase 4 shown as the combined duration of 4a and 4b; 95% conf. diff. lation changes for the Ardley Island penguin colony compared with a summary of key environmental inﬂuences g \| p g p p g y p g y p y y during the Holocene. (a) Holocene relative sea level (RSL) changes for the South Shetland Islands (SSI)45 in metres above present mean sea level (m a.p.s.l.). (b) Modelled Ardley Lake (16 m a.p.s.l.) penguin colony population changes in guano phases GP-1 to GP-5 (this study) with major colony impact eruptions (circles), and post-eruption recovery intervals (red stipple); estimated modelled population combined errors for the 0.96% and 11% guano- delivery models are not shown for clarity, but typically 30–50% (Supplementary Fig. 19). (c) Sub-fossil penguin occupation record from the Antarctic Peninsula (AP) based on radiocarbon dates of remains at abandoned nesting sites (refs 10,12,53): 1 ¼ Ardley Lake colony (this study), 2 ¼ SSI and the Northwest AP, 3 ¼ Northeast AP, 4 ¼ Mid-southern AP (Supplementary Data 5). (d) Summarized timing of warmer phases and periods of greater open water around the SSI and the AP48,29,56 over the last 10,000 years, deﬁned as 46–0 ka upper bound on their respective means (95% conﬁdence level), where: 5 ¼ Yanou Lake mean summer air temperature (MSAT) (this study), 6 ¼ Anvers Trough open water record (this study), 7 ¼ Palmer Deep sea surface temperature (SST)48, 8 ¼ James Ross Island temperature anomaly29, 9 ¼ AP warm periods56 (Supplementary Data 4). (e) Legend. ARTICLE (e) Palmer Deep (PD) Sea Surface Temperature (SST) 100-year interval records integrated over 0–200 m depth48 and at the surface47 with data 46–0 ka upper bound on the mean (95% conﬁdence level) shaded in dark red; (f) James Ross Island (JRI) ice core temperature anomaly record (compared to 1961–1990 CE interval) with s.e. ranges of the isotope-temperature dependence shown as grey lines29. (g) AP regional palaeoclimate synthesis56 (Supplementary Data 4). 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 1 4 5 3 2 15 m 10 m 5 m 2 3 4 1 +Warm 0–200 m +Warm surface a c d RRR MCA? EHO MHH 8 7 6 5 9 T5a T7 T4 T3b Modelled penguin population (log scale) b 0 1 100 10,000 ,Min Max Warmer Colder Colder e Open water >6–0 ka upper bound (95%) >6–0 ka upper bound (95%): Discontinuous warmth >6–0 ka upper bound (95%): Sustained warmth >2x 6–0 ka upper bound (95%): Sustained warmth AP Composite warm periods Volcanic eruptions Palaeoenvironmental data Penguin data VEI>4 VEI>3 VEI< or =3 Weighted mean eruption age and 95% min.–max. (cal a BP) Ardley Lake post-eruption colony recovery interval ARD guano phases GP-1 to GP-5 ARD exceptionally elevated guano phases ARD catchment deglaciation Published bone and egg ages (cal a ± 1σ) Modelled ARD bone ages (cal a ± 95% min.–max.) ARD bone collagen ages (cal a ± 1σ) Colony population modelling 11% Guano-delivery (most likely minimum) 0.96% Guano-delivery (most likely maximum) 100% Guano-delivery (unlikely) Ellipsoid basin (sediment-focussed); <30° slope accum. area RSL (m a.p.s.l.) 0 8 16 gure 5 \| Modelled penguin population changes for the Ardley Island penguin colony compared with a summary of key environmental inﬂuences e NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Discussion percentage of guano in the sediments, while the T6 eruption c. 6.0–5.9 cal ka BP [6.4–5.7 cal ka BP max.–min. 95% conﬁdence interval age range] occurred c. 200 years after the guano phase GP-1 decline had begun (Fig. 4a,b, Supplementary Note 9, Supplementary Discussion, Supplementary Table 3). Eruptions in the early Holocene (T7) and in the last c. 2,000 years (T1,T2) did not have a measurable impact on the Ardley Lake penguin colony as the populations were already at, or near, minima during these times. percentage of guano in the sediments, while the T6 eruption c. 6.0–5.9 cal ka BP [6.4–5.7 cal ka BP max.–min. 95% conﬁdence interval age range] occurred c. 200 years after the guano phase GP-1 decline had begun (Fig. 4a,b, Supplementary Note 9, Supplementary Discussion, Supplementary Table 3). Eruptions in the early Holocene (T7) and in the last c. 2,000 years (T1,T2) did not have a measurable impact on the Ardley Lake penguin colony as the populations were already at, or near, minima during these times. Ardley Lake began accumulating sediments c. 8.8 cal ka BP [9.2–8.4 cal ka BP] following retreat of the SSI Ice Cap from this part of the Fildes Peninsula between 10.1 and 8.2 cal ka BP (ref. 52), driven by early Holocene thermal maximum conditions recorded in regional marine and ice core temperature records (the early Holocene Optimum (EHO) in Fig. 4g (refs 29,47,48)). The highly explosive, T7 eruption, c. 7 cal ka BP [6.0–7.7 cal ka BP], had a bi-modal basaltic-andesitic and trachydacitic-rhyolitic NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Table 2 \| Major mid-late Holocene eruptions of the Deception Island volcano and Ardley Lake penguin colony recovery interval summary statistics. Volcanic eruption data Guano phase data Tephra layer Eruption size ARD & YAN Age 95% max.–mean (cal a BP) Decline to Fo.sed. o10% Recovery guano phase Recovery to Fo.sed. 410% Weighted mean Guano phase ended Weighted mean age [95% max.–min.] (cal a BP) Weighted mean age [95% max.–min.] (cal a BP) Recovery interval (years±95% conf. Discussion Warmer conditions have also been recorded at this time in lake and moss peat bank records from Livingston Island, Elephant Island, Beak Island56,58, some marine records from the AP48,56,59, Antarctic ice core composite records60 and stacked Southern Hemisphere temperature records61 (Fig. 4e–g, Supplementary Fig. 17e–i). These ‘more favourable’ climatic conditions for rearing juvenile penguins likely drove the Ardley Lake penguin colony to its Holocene population maxima, and led to the re-establishment of colonies in the mid-southern Peninsula region (Fig. 5c). Despite the gentoo penguins’ preference to feed near their colony, it appears that GP-4 was not adversely affected by the persistence of sea-ice in Maxwell Bay (Fig. 4c) and around the Anvers Shelf (Fig. 4d) and the cooler sea surface anomalies at PD (Fig. 4e). However, as with guano phases GP-2 and GP-3, guano phase GP-4 was abruptly terminated by a Deception Island eruption event (T3b). The peak in penguin populations during guano phase GP-1 (6.7–6.3 cal ka BP) coincided with a continuation of the local open water conditions in Maxwell Bay (Fig. 4c), but at the same time there were contrasting reductions in shelf-edge spring/ summer open water conditions 6.7–6.0 cal ka BP in the Anvers Trough (inferred from higher Fragilariopsis curta/F. kerguelensis diatom ratios and lower percentage of pelagic open water diatoms), and other records of ‘cooler’ and increased ‘sea-ice’ conditions around the SSI 7.3–5.2 cal ka BP (ref. 54). Local sea-ice conditions appear to be the most signiﬁcant factor in driving GP-1, which ended when there was a gradual increase in sea-ice in Maxwell Bay more than 200 years before the T6 eruption event. y y The following two (of three) prominent Ardley Lake guano dry mass accumulation maxima occurred during GP-2 and GP-3 c. 5.8–5.3 and 4.5–4.3 cal ka BP (Fig. 4b, Supplementary Table 7). In marked contrast to GP-1, both GP-2 and GP3 occurred during a period of greater sea-ice extent or seasonality in Maxwell Bay (Fig. 4c), despite the preference of gentoo penguins for ice-free conditions. These maxima also show no strong or consistent relationships with sea-ice conditions on the Anvers Shelf (Fig. 4d), ocean temperatures at PD (Fig. 4e) or atmospheric temperatures in the JRI ice core (Fig. 4f), although the peak of GP-3 corresponds with the onset of a ‘mid Holocene Hypsithermal’ (MHH in Fig. 4g) at c. 4.5 ka in several terrestrial cross-Peninsula records55,56. Discussion populations increasing during volcanically inactive periods and then experiencing abrupt catastrophic declines following major eruptions (Fig. 4b). p g After the T5a Deception Island eruption abruptly ended guano phase GP-2, the Ardley Lake colony struggled to fully re-establish itself for c. 800 years (Table 2) due to a series of closely spaced eruption events (Fig. 4b) and a phase of continued sea-ice presence in Maxwell Bay (Fig. 4c). Our analysis of longer-term (100-year interval) trends in the Maxwell Bay sea-ice reconstruc- tion52 (Fig. 4c) show that these cooler oceanographic conditions persisted well into the late Holocene (5.9–2.6 ka), corresponding to ‘cooler’ conditions and increased sea-ice in the Bransﬁeld Strait between c. 5 and 2 cal ka BP (ref. 57), and advancing glacier margins, increased local snow/ice and sea-ice and limited primary production in surface waters of King George Island54. Despite these cooler conditions the penguin population was able to re- establish itself during GP-3, but was then abruptly terminated by the T4 Deception Island eruption at c. 4.5–4.2 cal ka BP. Following the EHO, an extended period of generally ‘warmer’ interglacial conditions, with minor variations in temperature existed on the AP between 9.2 and 2.5 cal ka BP (refs 48,29) (Fig. 4f,g). These conditions, together with the extended periods of open water in Maxwell Bay (Fig. 4c) and on the Anvers Shelf (Fig. 4d) enabled the establishment of a small penguin colony on Ardley Island from c. 6.7 cal ka BP [7.4–5.8 cal ka BP]. This is the oldest in situ evidence of a Holocene penguin colony on the AP, and up to c. 1,000 years before the sub-fossil evidence of occupation for the mid-southern western AP from c. 5.8 cal ka BP [5.9–5.6 cal ka BP], and the northern AP from c. 5.5 cal ka BP [5.6–5.3 cal ka BP] (refs 12,53) (Fig. 5). p p The most sustained guano phase, GP-4 (c. 4.0–3.0 cal ka BP) (Table 1), occurred during the warm conditions of the late MHH (Fig. 4g), and is characterized by a marked increase in MSAT reconstructed in the Yanou Lake GDGT record (Fig. 4a), positive temperature anomalies at JRI (Fig. 4f) and a sustained period of signiﬁcantly warmer cross-Peninsula terrestrial temperatures from c. 3.8 cal ka BP (refs 29,48) (Fig. 4a,f,g, Supplementary Tables 7,8). Discussion diff.) [min.–max.] T5a VEI44 5,530–5,360 2 5,280 [5,430–5,160] 3 4,500 [4,630–4,300] 780±50 [530–1,130] T4 VEI43 4,480–4,230 3 4,310 [4,500–4,090] 4 3,900 [4,090–3,740] 410±30 [0–760] T3b VEI43 3,220–2,990 4 2,980 [3,090–2,860] 5 2,490 [2,700–2,160] 480±60 [160–390] Only eruptions that had a signiﬁcant impact on colony population levels are shown, where signiﬁcant is deﬁned as a reduction in the proportion of guano-inﬂuenced sediment (% Fo.sed.) to o10% within 200 years after an eruption event; the combined ARD and YAN 95% max. mean age was used to determine the most likely eruption age and is the maximum 95% conﬁdence age weighted mean age, derived from Bayesian age-depth modelling (see Supplementary Notes 5–7, Supplementary Methods); cal a BP is calibrated (or calendar) years before present, where BP is deﬁned as 1950 CE (Supplementary Methods); [95% max.- min.] is the maximum and minimum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling; weighted mean recovery interval duration (cal years) ¼ weighted mean decline age - weighted mean recovery age; 95% conf. diff. is the average difference between the weighted mean recovery interval and its respective 95% max. or min. decline max. or min. recovery interval (cal years); [min.- max.] is the absolute minimum to maximum recovery interval duration (cal years), where the minimum recovery interval duration ¼ 95% min. decline - 95% max. recovery age and the maximum recovery interval duration ¼ 95% max. decline 95% min. recovery age; the 95% conf. diff. weighted average recovery interval duration (±s.e.) ¼ 570±110 cal years (1s±160 cal years; 95% lower-upper conﬁdence interval ¼ 90–1050 cal years; signiﬁcance level a ¼ 0.05; age data are rounded to the nearest 10 years, but rounding to the nearest 100 years provides a more realistic assessment of age-depth modelling errors and comparison to 100-year interval data in Figs 4 and 5. See Supplementary Table 3 for ARD and YAN tephra deposits age data. Table 2 \| Major mid-late Holocene eruptions of the Deception Island volcano and Ardley Lake penguin colony recovery interval summary statistics. Guano phase data composition, and dispersed tephra widely across the northern Peninsula and Scotia Sea (Supplementary Figs 20,21). This and other more widespread early Holocene eruptions occurred before the Ardley Lake colony was established, and could have disrupted early post-deglaciation penguin colonization across the north-western AP (Supplementary Discussion). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 This resulted in an increase in the available area for nesting sites near the coast and may have driven the relocation of penguins from the Ardley Lake catchment to the western side of Ardley Island, where much of the present day colony is located (Fig. 6c). p y y g Although our new lake records suggest that warmer local atmospheric temperatures (Figs 4a,5) and the regional expression of the MHH led to increased penguin populations during guano phase GP-3 c. 4.5–4.3 cal ka BP and GP-4, c. 4.0–3.0 cal ka BP, our main ﬁnding is that large mid-late Holocene eruptions of the Deception Island volcano had a sporadic, but devastating, impact on the Ardley Lake penguin colony. All ﬁve phases of colony expansion occurred in the absence of large volcanic eruptions and three of the phases (GP-2, 3, 4) were abruptly ended by eruptions from the Deception Island volcano at c. 5.5–5.4 cal ka BP (T5a), 4.5–4.2 cal ka BP (T4) and 3.2–3.0 cal ka BP (T3b). Only guano phase GP-5 declined gradually, which we attribute to adverse ‘neoglacial’ conditions and migration away from nesting sites in the centre of the island towards newly emergent coastal areas. This guano phase appears to have been negatively impacted by the relatively minor T2 eruption event at c. 2.1 cal ka BP, but, in general, from c. 2,500 years onwards the Ardley Lake colony was too small to determine whether volcanic activity had a signiﬁcant impact. ( ) No signiﬁcant changes in the Ardley Lake colony have occurred in the last c. 1,000 years (Fig. 4), although we detected a minor increase in guano input into Ardley Lake from c. 500 years onwards (Fig. 5), which coincides with the onset of warming detected in north-eastern AP lake58 and ice core records29 from c. 500 years, after the northern AP ‘neoglacial’ minimum. Elsewhere, increases in guano input into Lake Y2 on Ardley Island (Fig. 6) and in Hope Bay records from the north- eastern AP10,16 have been linked to ‘warming’ associated with a ‘Medieval Climate Anomaly’43, inferred in some AP records between c. 1,200 and 600 years ago48,56,66. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 5a). After deglaciation, during the Early Holocene Warm Optimum (EHO) (11.5–9.5 ka)56, the land area available on Ardley Island was c. 0.6 km2, c. 30–35% less than the present day. The eastern half of the island, where the current penguin colony is located, was bordered by steep cliffs forcing early to mid Holocene colonies to nest in the centre of Ardley Island. During the mid to late Holocene, relative sea level (RSL) fell, increasing the land area available. The amount of guano deposited in Ardley Lake declined after c. 1,300 cal a BP as some colonies relocated to the Lake Y2 and Lake G catchments43. The eastern side of the island became more easily accessible when RSL fell below 5 m above present sea level (m a.p.s.l.) after c. 1,300 years ago. Future colony population increases could be accommodated in the central area of Ardley Island. See Supplementary Fig. 1 and Supplementary Data 6 for modern-day penguin population data; see Supplementary Fig. 22 for an extended version of this ﬁgure. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. BP. This ‘neoglacial’ is apparent in Peninsula-wide lake58, marine57,59 and ice core29 records (Fig. 4f), and marked by the episodic re-advance of local glaciers52,62 that slowed the rate of RSL decline on the SSI63,64. Although reconstructed temperatures in the YAN-GDGT record remained above average until c. 1.5 cal ka BP, sea-ice conditions in Maxwell Bay and the Bransﬁeld Strait52,57, and at some localities further south65, became progressively less favourable between c. 2.3 and 1.8 cal ka BP (ref. 10), likely constraining the growth of the Ardley Lake and Lake Y2 colonies. The relatively minor (VEIo3) T2 eruption event had a measurable impact on the Ardley Lake colony in the middle of guano-phase GP-5, c. 2.1 cal ka BP (Fig. 4b), but further deterioration in north-western AP climate into the ‘neoglacial’ minimum probably drove more progressive reductions in the Ardley Lake GP-5 and Lake Y2 colonies from c. 1.5 to 1.3 cal ka BP (ref. 43). Y2 sites during the last c. 1,000 years mirrors the increased rate of RSL fall from c. 10 to 0 m a.p.s.l. (refs 45,64,70) (Fig. 6). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Discussion Instead, the main driver of these penguin population changes is volcanism, with colony We link the failure to re-establish another large and sustained colony of similar size and duration as guano phase GP-4 to the start of a marked ‘neoglacial’ decline in temperature at c. 2.5 cal ka 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Y2 G Y4 b 4,200–1,300 years ago <15–10 m a.p.s.l Ardley Lake <15–10 m a.p.s.l b 4,200–1,300 years ago b 4,200–1,300 years ago >15 m a.p.s.l. 9,000–4,200 years ago a 9,000–4,200 years ago a AD4 AD3 13.4–15 m 10.5–11.1 m 4.5–6.6 m Y2 Y4 G Raised beaches c <10–0 m a.p.s.l. 1,300 years ago–present day Ardley Lake d Main penguin landing sites Secondary penguin landing sites Penguin population >1,000 0 a b RSL (m a.p.s.l.) 0 16 0 2,000 4,000 6,000 8,000 10,000 c Age (cal a BP) d d c 1,300 years ago–present day c Ardley Lake Figure 6 \| Changes in land availability and colony population on Ardley Island over the last 9,000 years. (a) 9,000 to 4,200 years ago; (b) 4,200 to 1,300 years ago; (c) 1,300 years ago to the present day; (d) Legend and relative sea level (RSL) scenarios shown in (a)–(c) (see Fig. 5a). After deglaciation, during the Early Holocene Warm Optimum (EHO) (11.5–9.5 ka)56, the land area available on Ardley Island was c. 0.6 km2, c. 30–35% less than the present day. The eastern half of the island, where the current penguin colony is located, was bordered by steep cliffs forcing early to mid Holocene colonies to nest in the centre of Ardley Island. During the mid to late Holocene, relative sea level (RSL) fell, increasing the land area available. The amount of guano deposited in Ardley Lake declined after c. 1,300 cal a BP as some colonies relocated to the Lake Y2 and Lake G catchments43. The eastern side of the island became more easily accessible when RSL fell below 5 m above present sea level (m a.p.s.l.) after c. 1,300 years ago. Future colony population increases could be accommodated in the central area of Ardley Island. See Supplementary Fig. 1 and Supplementary Data 6 for modern-day penguin population data; see Supplementary Fig. 22 for an extended version of this ﬁgure. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. Figure 6 \| Changes in land availability and colony population on Ardley Island over the last 9,000 years. (a) 9,000 to 4,200 years ago; (b) 4,200 to 1,300 years ago; (c) 1,300 years ago to the present day; (d) Legend and relative sea level (RSL) scenarios shown in (a)–(c) (see Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 AD4 AD3 13.4–15 m 10.5–11.1 m 4.5–6.6 m Y2 Y4 G Raised beaches ? ? ? Y2 G Y4 Y4 9,000–4,200 years ago a b c d 4,200–1,300 years ago <10–0 m a.p.s.l. >15 m a.p.s.l. <15–10 m a.p.s.l 1,300 years ago–present day Main penguin landing sites Secondary penguin landing sites Penguin population >1,000 0 a b RSL (m a.p.s.l.) 0 16 0 2,000 4,000 6,000 8,000 10,000 c Ardley Lake Ardley Lake Ardley Lake Age (cal a BP) Figure 6 \| Changes in land availability and colony population on Ardley Island over the last 9,000 years. (a) 9,000 to 4,200 years ago; (b) 4,200 to 1,300 years ago; (c) 1,300 years ago to the present day; (d) Legend and relative sea level (RSL) scenarios shown in (a)–(c) (see Fig. 5a). After deglaciation, during the Early Holocene Warm Optimum (EHO) (11.5–9.5 ka)56, the land area available on Ardley Island was c. 0.6 km2, c. 30–35% less than the present day. The eastern half of the island, where the current penguin colony is located, was bordered by steep cliffs forcing early to mid Holocene colonies to nest in the centre of Ardley Island. During the mid to late Holocene, relative sea level (RSL) fell, increasing the land area available. The amount of guano deposited in Ardley Lake declined after c. 1,300 cal a BP as some colonies relocated to the Lake Y2 and Lake G catchments43. The eastern side of the island became more easily accessible when RSL fell below 5 m above present sea level (m a.p.s.l.) after c. 1,300 years ago. Future colony population increases could be accommodated in the central area of Ardley Island. See Supplementary Fig. 1 and Supplementary Data 6 for modern-day penguin population data; see Supplementary Fig. 22 for an extended version of this ﬁgure. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. Y4 9,000–4,200 years ago a >15 m a.p.s.l. Ardley Lake ? ? ? Y2 G Y4 b d 4,200–1,300 years ago <15–10 m a.p.s.l Main penguin landing sites Secondary penguin landing sites Penguin population >1,000 0 a b RSL (m a.p.s.l.) 0 16 0 2,000 4,000 6,000 8,000 10,000 c Ardley Lake Age (cal a BP) ? ? ? Methods S l ll Sample collection and laboratory analyses. Sediment cores extracted from Ardley Lake (ARD) and Yanou Lake (YAN) were analysed for the concentration of elements associated with changing inputs of penguin guano and volcanic ash deposits using complementary multi-proxy biogeochemical techniques described in this section and in Supplementary Methods. Chronologies for the lake sediment cores were established using 18 (ARD) and 15 (YAN) AMS radiocarbon (14C) ages from, in order of preference: (1) moss macrofossil layers (consisting of hand-picked ﬁne strands of the aquatic moss Drepanocladus longifolius (Mitt.) Paris, but also occasional layers of Campylium polygamum (Schimp.) Lange & C.E.O. Jensen, and some unidentiﬁable/mixed species moss fragments—considered more likely to have been reworked; (2) terrestrial and/or lacustrine algae; (3) other intact macrofossils and sub-fossils, including bones (bone-collagen, where extractable); (4) other (macro)fossil fragments; (5) organic-rich bulk sediments and, near the base of each core and as a last resort, (6) bulk glaciolacustrine or glaciomarine sediments (Supplementary Table 2). Bulk sediments were only dated where macrofossils were absent, while paired macrofossil or bone-collagen and bulk sediment samples were measured in the surface sediment and wherever present to check for any systematic offsets between ages obtained from different carbon sources (see Supplementary Methods, Supplementary Notes 5 and 6 for more details). ) Physical disablement and poisoning are the most signiﬁcant potential hazards to penguins, even though they are well-adapted to elevated levels of F, Cu, Cd, and Hg through high dietary intake16. Prolonged exposure to the ﬁne abrasive glass particles in tephra while rearing chicks on land, or during feeding at sea, could adversely affect one (or all) of the major physiological processes of penguins and their prey (for example, respiration, digestion, immune function, vision; Supplementary Discussion). Colonies are more likely to be decimated if signiﬁcant amounts of ash fell during the breeding season onto unhatched eggs or recently hatched chicks, or if one parent failed to return from foraging. Mature individuals on long forages, outside the areas directly affected by ashfall, would have the best chance of survival. Our recovery interval calculations suggest that these individuals may have been displaced to alternative nesting sites such as those that were occupied in the mid-southern AP at c. 5.4 and 4.2 cal ka BP (Fig. 5c). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 The increase in sub- fossil evidence of nesting sites across the AP in the last 1,000 years for all Pygoscelis spp., regardless of whether they are ‘ice-dependent’ or ‘ice-avoiding’ species, could be a response to the more favourable ‘Medieval Climate Anomaly’ and/or post-‘neoglacial’ conditions, or reﬂect migratory shifts to newly emerged, low-lying coastal areas created by declining Holocene RSLs across the Peninsula63,64,67–69 (for example, Fig. 5a, c). The absence of signiﬁcant colonies at both the Ardley Lake and Lake The remoteness of active volcanism in Antarctica means that modern-day analogues for volcanic impacts on penguin colonies are inevitably sparse and hard to quantify. Although the relative size of Deception Island eruptions is thought to have diminished in the late Holocene, comparatively small recent eruptions were locally hazardous, causing catastrophic ﬂooding and lahars20. Larger mid-late Holocene eruptions are thought to have generated pyroclastic density ﬂows and surges20,21, which could have been particularly destructive at ground level. Circumstantial evidence from the volcanically active South Sandwich Islands 10 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 shows that, close to an erupting volcano, penguins are largely absent from areas affected by recent ash fall (Supplementary Fig. 23). Proximal fallout of coarse-grained tephra could lead to high localized mortality rates and signiﬁcantly disrupt nesting activities, or drive migration to areas less affected by ash. The northern AP is deﬁned as having a ‘moderate to extremely high likelihood of supraglacial airfall by active volcanoes’, where a ‘signiﬁcant risk to life’ exists31, mainly because the deposition of far-travelled ﬁne black basaltic ash can lead to rapid snow-melt and (supraglacial) lahar formation71. Other indirect processes, such as burial by tephra, the generation of tsunamis, landscape destabilization, and the deposition of ﬁne ash layers and aerosols could pose a substantial risk to distal penguin colonies30, such as those on Ardley Island (Supplementary Fig. 12, Supplementary Discussion). nesting and foraging activities on Ardley Island. Moreover, the saw-toothed, asymmetric pattern of successive Ardley Lake penguin colony population maxima suggests that sustainable colony recovery following these mid-late Holocene volcanic eruptions was slow, taking, on average, between 400 and 800 years, but possibly as much as 1,100 years during the most disruptive phase of volcanism (T5) c. 5.5–4.6 cal ka BP. Methods S l ll For data replication and comparison purposes, the total inorganic carbon (TIC) of 69 (out of 385) samples from both cores was determined coulometrically by a CM 5012 CO2 coulometer coupled to a CM 5130 acidiﬁcation module (UIC, USA) while total organic carbon was then calculated as the difference between TC and TIC (TOC% ¼ TC% TIC%). Owing to the high correlation between TC and TOC (R240.9997) and the negligible TIC concentrations (av. 0.10 and o0.01 mass%) compared to TC values (av. 6.2 and 2.5 mass%) in both cores, TC is considered to reﬂect the amount of organic carbon in both lake sediments. Quantitative XRF analysis for major and trace elements (SiO2, Al2O3, CaO, K2O, Na2O, P2O5, As, Ba, Cu, Co, Ni, Sr, Y, Zn, Zr) was carried out with a conventional wavelength dispersive X-ray ﬂuorescence (WD-XRF) spectrometer (Philips PW 2400). For WD-XRF, glass beads were prepared following standard procedures74 and measurements undertaken in random order to avoid artiﬁcial trends. Trace element analysis (Cd, REE) of selected samples was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Element 2 mass spectrometer, Thermo Scientiﬁc, Germany) at 2,500-fold dilution. For additional details concerning ICP-MS measuring conditions, see Schnetger75. Selenium was determined on acid digestions by graphite atomic absorption spectrometry (G-AAS) using a Unicam 939 QZ AA spectrometer and a Zeeman- effect background correction. A Milestone DMA-80 Direct Mercury Analyser was used for the measurement of mercury via cold vapour atomic absorption spectroscopy (CV-AAS). ICP-MS and G-AAS standard acid digestion procedures are described in Supplementary Methods. F ll i t bli h d d d i l d i ti lid d pp y Sub-samples for carbon, nitrogen, XRF and ICP-MS (quantitative, dry mass) bio-element analyses were taken at 1 cm intervals from ARD and YAN lake sediment cores, lyophilized and ground with an agate ball mill and manually with an agate pestle and mortar. ARD samples were analysed for total carbon (TC), total sulphur (TS) and total nitrogen (TN) using a CNS analyser (vario EL Cube, Elementar, Germany) equipped with a solid-state infrared and a heat conductivity detector. TC and TS measurements were conducted on YAN sediments by means of an ELTRA CS analyser. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Methods S l ll The physical landscape disturbance and presence of tephra over Ardley Island prevented sustainable post-eruption reoccupation of the site for, on average, 570±110 years (weighted mean±s.e.), but as much as c. 800–1,100 years following the largest mid-late Holocene (T5a) eruption c. 5,500 years ago (Table 2). Measured radiocarbon ages from samples shown in Supplementary Table 2 were calibrated using SH13 and MARINE13 calibration curves and age-depth models were generated using Bayesian age-depth modelling techniques (Fig. 2, Supplementary Figs 3,9, Supplementary Methods and Supplementary Notes 5–7). All the ‘as measured’ (uncalibrated) radiocarbon age data shown in Supplementary Table 2 were used as input data for ﬁnal age-depth model runs (ARD-M5, YAN-M4, ANVERS-M1, where model run number is indicated by the -M sufﬁx). The weighted mean basal age of the ARD core was 8,750 cal a BP [8,410–9,230 min –max. 95% conﬁdence age range]. Error analysis shows that whole record mean 95% conﬁdence age-depth model uncertainties, rounded to the nearest 10 years with 95% conﬁdence minimum to maximum age ranges (in years at depths in cm) shown in square brackets are: ARD-M5: 410 years [0.6 years at 0 cm–1,430 years at 335 cm], YAN-M4: 600 years [160 years at 7.7 cm–1,990 years at 340 cm], and ANVERS-M1: 1,420 years [590 years at 0 cm–1,810 years at 375 cm]. Equivalent values for the late Holocene in each record are: ARD-M5: 640 years [0.6 years at 0 cm–750 years at 20 cm], YAN-M4: 680 years [160 years at 7.7 cm–760 years at 2.7 cm] and ANVERS-M1: 1,370 years [590 years at 0 cm–1,600 years at 155 cm] (Supplementary Note 7). Measured radiocarbon ages from samples shown in Supplementary Table 2 were calibrated using SH13 and MARINE13 calibration curves and age-depth models were generated using Bayesian age-depth modelling techniques (Fig. 2, Supplementary Figs 3,9, Supplementary Methods and Supplementary Notes 5–7). All the ‘as measured’ (uncalibrated) radiocarbon age data shown in Supplementary Table 2 were used as input data for ﬁnal age-depth model runs (ARD-M5, YAN-M4, ANVERS-M1, where model run number is indicated by the -M sufﬁx). The weighted mean basal age of the ARD core was 8,750 cal a BP [8,410–9,230 min –max. 95% conﬁdence age range]. Methods S l ll Error analysis shows that whole record mean 95% conﬁdence age-depth model uncertainties, rounded to the nearest 10 years with 95% conﬁdence minimum to maximum age ranges (in years at depths in cm) shown in square brackets are: ARD-M5: 410 years [0.6 years at 0 cm–1,430 years at 335 cm], YAN-M4: 600 years [160 years at 7.7 cm–1,990 years at 340 cm], and ANVERS-M1: 1,420 years [590 years at 0 cm–1,810 years at 375 cm]. Equivalent values for the late Holocene in each record are: ARD-M5: 640 years [0.6 years at 0 cm–750 years at 20 cm], YAN-M4: 680 years [160 years at 7.7 cm–760 years at 2.7 cm] and ANVERS-M1: 1,370 years [590 years at 0 cm–1,600 years at 155 cm] (Supplementary Note 7). ( ) In conclusion, while we cannot assess some factors that determine breeding success such as pressure from predators1, the ability to change diet72, relative changes in species composition, or recent anthropogenic impacts73, our detailed case study, using innovative methodologies, has revealed the key environmental factors inﬂuencing the Ardley Lake penguin colony through the Holocene. Once deglaciated, c. 8,500 years ago, climatic conditions on Ardley Island were sufﬁciently amenable throughout the rest of the Holocene for sustained penguin habitation. The Ardley Lake colony population maximum in ﬁrst half of the late Holocene, c. 4,000 to 3,000 years ago, occurred during a period of particularly ‘favourable’ regional climate (sustainably warmer than the 6–0 ka average, with fewer storms) and local-regional sea-ice conditions (near-average 6–0 ka sea-ice cover/seasonal extent), which enhanced nesting and foraging success. Conversely, ‘neoglacial’ conditions and falling sea levels during the second half of the late Holocene (c. 2,000 years ago to present) contributed to colony decline and/or migration away from the centre of Ardley Island. ( pp y ) Sub-samples for carbon, nitrogen, XRF and ICP-MS (quantitative, dry mass) bio-element analyses were taken at 1 cm intervals from ARD and YAN lake sediment cores, lyophilized and ground with an agate ball mill and manually with an agate pestle and mortar. ARD samples were analysed for total carbon (TC), total sulphur (TS) and total nitrogen (TN) using a CNS analyser (vario EL Cube, Elementar, Germany) equipped with a solid-state infrared and a heat conductivity detector. TC and TS measurements were conducted on YAN sediments by means of an ELTRA CS analyser. ARTICLE The target ions were m/z 1,302, 1,300, 1,298, 1,296 and 1,292 for the isoprenoid GDGTs (isoGDGTs) compounds and 1,050, 1,048, 1,046, 1,036, 1,034, 1,032, 1,022, 1,020 and 1,018 for the branched GDGT (brGDGTs) za ¼ zm 2 3 z : zm ð Þ0 =3½2 z : zm ð Þ0 1 Units : cm ð3Þ ð3Þ For a hyperboloid basin shape: SGðtÞhyper ¼ 2 3 1 z2 m 2zm z ð Þ2 dzG dtG Units : g cm 2a 1 ð4Þ where z ¼ core depth at time t; zm ¼ maximum depth of the original basin (that is, current lake depth þ sediment record depth); z : zm ð Þ0 ¼ ratio of the mean depth ðzÞto the maximum depth of the original basin (zm); dzG=dtG ð Þ ¼ guano dry mass accumulation rate ¼ Fo.sed DMAR in g cm 2 a 1. g Assuming Ardley Lake best approximates to an ellipsoid basin shape (that is, the ‘more likely’ basin shape scenario), the total basin-wide guano accumulated (IG) over the ‘more likely’ o30 degree slope accumulation area (Ao30) within Ardley Lake (Supplementary Fig. 2), corrected for the effects of sediment focussing, is then: Diatom analysis of the ARD and YAN records is as described in ref. 45 and summarized in Supplementary Fig. 14, while Anvers Shelf marine core (ANVERS) diatom analysis and chronology are described in Supplementary Methods and summarized in Supplementary Fig. 15. IGAo30ellipðtÞ ¼ SGðtÞellip: Ao30 ½ Units : g cm 2a 1cm2 ¼ g a 1 ð5Þ Data analysis. Bio-element assemblages are immobile in (Antarctic) lake sedi- ments43 and guano input to sediments leads to the formation and preservation of stable phosphates, such as struvite (Mg(NH4)PO4 x 6 H2O), leukoposphite and, in particular, hydroxylapatite (Ca5(PO4)3(OH)), which is one of the dominant compounds found in ornithogenic soils/sediments on King George Island. During the precipitation of apatites, an exchange between Ca2 þ, PO43 , F and OH and elements such as Ag, Br, Ba, Cd, Cu, Cr, I, Na, Mg, Mo, Pb, S, Se, Sr, U, V, Y and Zn is possible, coupled to microbial-mediated degradation of solid phases78 (see Supplementary Note 1 for more details). ARTICLE Contiguous 1 cm subsamples were taken in the top 20 cm of the YAN record, and at 2 cm intervals between 20 and 38 cm and 188 and 210 cm core depth (dated to c. 6.1 cal ka BP). Freeze dried and homogenized sediment samples weighing 0.2–4.3 g were microwave-extracted in DCM:Methanol (3:1, v/v). The total extracts were saponiﬁed and GDGTs isolated following ref. 76. Prior to analysis the GDGT extracts were ﬁltered through a 0.2 mm Whatman PTFE ﬁlter. GDGT analysis was undertaken using an Acquity Xevo TQ-S (triple quadrupole with step wave; Waters Ltd.) LC-MS set up with an atmospheric pressure chemical ionization source (Ion saber II) operated in positive ion mode. Analytical separation was achieved using a Grace Prevail Cyano HPLC column (3 mm, 150 2.1 mm i.d.) ﬁtted with an in-line ﬁlter (Waters Acquity UPLC in-line ﬁlter, 0.2 mm) at 40 C using a binary solvent gradient where eluent A was hexane and eluent B was propanol. The ﬂow rate of h b l h l h d ﬁl f To summarize, the basin-wide guano inﬂux rate, SG, at time t, and total Ardley basin maximum core depths of z (water depth þ sediment depth, in cm) at time t, was calculated as follows: For an ellipsoid-basin: SGðtÞellip ¼ 2 3 1 z2 a 2za þ z ð Þ2 dzG dtG Units : g cm 2a 1 ð2Þ ð2Þ the mobile phase was 0.2 ml per minute with a gradient proﬁle of 99% A 1% B (0–50 min); 98.2% A 1.8% B (50–55 min); 90% A 10% B (55–65 min) and ﬁnally 99% A 1% B (66–80 min). The LC-MS settings were: source offset 50 V, capillary and 1.5 kV, desolvation temperature 200 C, cone voltage 30 V, desolvation gas (N2). Detection was achieved using selected ion monitoring of targeted [M þ H] þ ions (dwell time 50 ms). ARTICLE The average relative proportion of guano or ornithogenically derived sediment in the Ardley Lake sediment matrix (Fo.sed.) was estimated by using the mixing equation (equation (1)), modiﬁed after Shultz and Calder80: Several studies have shown that catchment erosion and deposition in small closed-basins with a single catchment and non-complex inﬂow characteristics (for example, Ardley Lake) can be approximated by a linear relationship (see Several studies have shown that catchment erosion and deposition in small closed-basins with a single catchment and non-complex inﬂow characteristics (for example, Ardley Lake) can be approximated by a linear relationship (see Supplementary Note 8 for references); thus, a reasonable approximation of the total amount of sediment or, in this case, guano delivered (GD), by erosion (Ge), into the Ardley Lake basin, can be obtained, simply from ratio of lake-area to catchment- area, expressed as an estimated percentage: Fo:sed: rel:% ð Þ ¼ P El¼Cu;Sr;Zn;P El=Al ð Þsmp El=Al ð Þbgd El=Al ð Þo:sed: El=Al ð Þbgd 4 100 ð1Þ rel % ð Þ ¼ P El¼Cu;Sr;Zn;P El=Al ð Þsmp El=Al ð Þbgd El=Al ð Þo:sed: El=Al ð Þbgd ð1Þ example, Ardley Lake) can be approximated by a linear relationship (see Supplementary Note 8 for references); thus, a reasonable approximation of the total amount of sediment or, in this case, guano delivered (GD), by erosion (Ge), into the Ardley Lake basin, can be obtained, simply from ratio of lake-area to catchment- area, expressed as an estimated percentage: (El/Al)smp is the element/aluminium ratio of selected bio-elements (Cu, P, Sr, Zn) in Ardley Lake sediments and (El/Al)bgd (mean Ardley Island bedrock51) and (El/ Al)o.sed. (guano-bearing ornithogenic sediment and soils49) represent the respective ratios in both end members. In order to minimize misinterpretation of this proxy, we calculated the average fraction of ornithogenic sediment and soils in the lake sediments using a combination of four chemically unrelated El/Al ratios. This provided a buffering effect on possible variations in ornithogenic soil and bedrock composition over time. In the resultant Fo.sed. percentage plotted in Fig. 4b, for example, Fo.sed. ¼ 20%, means that 20% of the sediment sample is eroded ornithogenic sediment-soil and 80% eroded bedrock. Theoretically, all Fo.sed. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 determine the precise position of tephra deposits (see Supplementary Methods, Supplementary Note 9, Supplementary Figs 3,7,10,12 for details). Electron Probe Micro-Analysis (EPMA) of 165 glass-shards from the most prominent T4–T7 tephra layers in the YAN, ARD and Beak 1 Lake records (Beak Island; Layers Ta–e (ref. 63)) was used to determine the eruption characteristics and source of mid-late Holocene eruptions that likely had the biggest environmental impacts. Results were compared with glass-shard analyses from age-equivalent tephra layers in marine cores PC460/461 from the Scotia Sea (new data; this study) and similarly analysed data in Moreton and Smellie23 from the Scotia Sea, northern Weddell Sea and Boyd Strait, and in Fretzdorff and Smellie22 from the Bransﬁeld Basin (see Supplementary Methods, Figs 20,21, Supplementary Table 10). 40% provide evidence for guano input above the local bedrock-terrigenous background level, but we used a weighted mean Fo.sed. value of 410% at the cut-off for CONISS-deﬁned geochemical zone to indicate the sustained presence of penguins around Ardley Lake (Table 1, Supplementary Table 7). We calculated post-eruption colony recovery intervals as the time taken until the percentage Fo.sed. value ﬁrst returned to 410% within the subsequent guano phase to represent the return of a sustained penguin presence around Ardley Lake. Measurement errors of o1% mean our deﬁnitions provide very conservative buffers of penguin presence during guano phases 1–5 (GP-1 to GP-5) (Table 2). Penguin population modelling results shown in Fig. 5 utilized dry mass Fo.sed.. accumulation rates (Fo.sed. DMAR in Fig. 4b), which were calculated following standard procedures81 (that is, multiplying Fo.sed. percentages by the dry mass accumulation rate, calculated from sedimentation rate and dry mass density data). We also used published penguin population parameters40,41 and the ‘most-realistic’ sediment focussed ellipsoid basin accumulation models81, assuming a o30 slope- angle accumulation area within Ardley Lake of 5,682 m2 (out of a maximum 7,274 m2 lake basin area) (see Supplementary Fig. 2). Further accumulation area scenarios, equations and references can be found in Supplementary Note 8. We considered the shape of Ardley Lake basin to approximate between a steep-sided hyperboloid basin and an ellipsoid basin (as below). Supplementary Methods, Figs 20,21, Supplementary Table 10). For the YAN-GDGT reconstructed temperature record, temperature-sensitive glycerol dialkyl glycerol tetraethers (GDGTs) biomarkers were extracted from 41 samples using a microwave-assisted solvent extraction76,77. ARTICLE These trace elements, which have naturally higher concentrations in penguin guano, are enriched in these phosphate phases and then immobilized in soils and sediment proﬁles during this substitution process. While the high correlation between Mg and P in ornithogenic soils from Vestfold Hills has been used to indicate the presence of struvite79, their lack of correlation in the Ardley Lake sediments means that Mg is likely derived from the bedrock lithology (Supplementary Fig. 3, Supplementary Table 4), and hydroxylapatite (Ca5(PO4)3(OH)) formation reﬂects guano input (Supplementary Fig. 6). where Ao30 ¼ 5; 682 568 m2 ¼ 5:68107cm2; SGðtÞellip ¼ the value for each 100-year interval obtained from equation (2). Using guano production rates of 84.5±21.1 g (25% error estimate applied) per penguin per day on Ardley Island, and a mean density of 0.31±0.19 gentoo penguins per m2 (for Signy Island)40,41, we estimated the total amount of guano delivered by erosion from the catchment into Ardley Lake as the guano yield (Gy) and the area required for the reconstructed population. As the main breeding season and ice/snow-free periods, when active erosion from catchments to lakes occurs on Ardley Island, last for approximately 3–4 months each year (91.3±22.8 days; 25% error estimate; equation (6) and Supplementary Table 9), we calculated the maximum amount of guano deposited per penguin (p 1), per year (a 1) inside the catchment and delivered to the lake as the maximum possible guano yield (Gy-100%) as follows: Gy100% ¼ D Pg ¼ 7; 716 2; 728 g a 1p 1 c:35% combined error ð Þ ð6Þ where D ¼ mean occupation time per year±estimated error ¼ 365.25/4 or 91.3±22.8 days (25% error applied); Pg ¼ amount, in grams (g) of guano deposited per year (a 1) per penguin (p 1). Methods S l ll For data replication and comparison purposes, the total inorganic carbon (TIC) of 69 (out of 385) samples from both cores was determined coulometrically by a CM 5012 CO2 coulometer coupled to a CM 5130 acidiﬁcation module (UIC, USA) while total organic carbon was then calculated as the difference between TC and TIC (TOC% ¼ TC% TIC%). Owing to the high correlation between TC and TOC (R240.9997) and the negligible TIC concentrations (av. 0.10 and o0.01 mass%) compared to TC values (av. 6.2 and 2.5 mass%) in both cores, TC is considered to reﬂect the amount of organic carbon in both lake sediments. Quantitative XRF analysis for major and trace elements (SiO2, Al2O3, CaO, K2O, Na2O, P2O5, As, Ba, Cu, Co, Ni, Sr, Y, Zn, Zr) was carried out with a conventional wavelength dispersive X-ray ﬂuorescence (WD-XRF) spectrometer (Philips PW 2400). For WD-XRF, glass beads were prepared following standard procedures74 and measurements undertaken in random order to avoid artiﬁcial trends. Trace element analysis (Cd, REE) of selected samples was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Element 2 mass spectrometer, Thermo Scientiﬁc, Germany) at 2,500-fold dilution. For additional details concerning ICP-MS measuring conditions, see Schnetger75. Selenium was determined on acid digestions by graphite atomic absorption spectrometry (G-AAS) using a Unicam 939 QZ AA spectrometer and a Zeeman- effect background correction. A Milestone DMA-80 Direct Mercury Analyser was used for the measurement of mercury via cold vapour atomic absorption spectroscopy (CV-AAS). ICP-MS and G-AAS standard acid digestion procedures are described in Supplementary Methods. Overall, though, we found no consistent relationships between guano inputs into Ardley Lake and local-regional atmospheric and ocean temperatures or sea-ice conditions across the Holocene. Instead, the overriding driver of long-term penguin colony change on Ardley Island was volcanic activity from Deception Island. Three out of ﬁve mid-late Holocene guano- phases were interrupted by volcanic ash fallout from eruptions that were at least an order of magnitude greater than present-day eruptions. Mid-late Holocene volcanic eruptions deposited layer of volcanic ash across the landscape, which severely disrupted Following established procedures, we used visual descriptions, smear slides and contiguous micro-XRF (m-XRF) scanning, at 200 mm and 2 mm intervals, to 11 ARTICLE Contiguous 1 cm subsamples were taken in the top 20 cm of the YAN record, and at 2 cm intervals between 20 and 38 cm and 188 and 210 cm core depth (dated to c. 6.1 cal ka BP). Freeze dried and homogenized sediment samples weighing 0.2–4.3 g were microwave-extracted in DCM:Methanol (3:1, v/v). The total extracts were saponiﬁed and GDGTs isolated following ref. 76. Prior to analysis the GDGT extracts were ﬁltered through a 0.2 mm Whatman PTFE ﬁlter. GDGT analysis was undertaken using an Acquity Xevo TQ-S (triple quadrupole with step wave; Waters Ltd.) LC-MS set up with an atmospheric pressure chemical ionization source (Ion saber II) operated in positive ion mode. Analytical separation was achieved using a Grace Prevail Cyano HPLC column (3 mm, 150 2.1 mm i.d.) ﬁtted with an in-line ﬁlter (Waters Acquity UPLC in-line ﬁlter, 0.2 mm) at 40 C using a binary solvent gradient where eluent A was hexane and eluent B was propanol. The ﬂow rate of the mobile phase was 0.2 ml per minute with a gradient proﬁle of 99% A 1% B (0–50 min); 98.2% A 1.8% B (50–55 min); 90% A 10% B (55–65 min) and ﬁnally 99% A 1% B (66–80 min). The LC-MS settings were: source offset 50 V, capillary 1.5 kV, desolvation temperature 200 C, cone voltage 30 V, desolvation gas (N2). Detection was achieved using selected ion monitoring of targeted [M þ H] þ ions (dwell time 50 ms). The target ions were m/z 1,302, 1,300, 1,298, 1,296 and 1,292 for the isoprenoid GDGTs (isoGDGTs) compounds and 1,050, 1,048, 1,046, 1,036, 1,034, 1,032, 1,022, 1,020 and 1,018 for the branched GDGT (brGDGTs) compounds. GDGTs were identiﬁed and integrated using MassLynx software (v.4.1) and GDGT-derived temperatures were calculated using the Antarctic and sub-Antarctic GDGT-temperature calibration77 (see Data Analysis section). The Antarctic and sub-Antarctic GDGT-MSAT surface calibration data set (comprising 32 sites in total) includes surface sediments from Yanou Lake, Ardley Lake and three other lakes from Fildes Peninsula, along with two further lakes from Potter Peninsula and four lakes from the Trinity Peninsula, north-eastern AP (including Beak Island). pp y g pp y For the YAN-GDGT reconstructed temperature record, temperature-sensitive glycerol dialkyl glycerol tetraethers (GDGTs) biomarkers were extracted from 41 samples using a microwave-assisted solvent extraction76,77. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 For Ardley Lake, the amount of guano delivered in grams (g) per year (a 1) per penguin (p 1) in the 11% guano (Gy–11%) yield scenario is given by: assess the uncertainties in cluster analysis. We used constrained incremental sum of squares (CONISS) stratigraphically constrained cluster analysis with broken stick analysis to deﬁne Diatom Zones (D) in the ARD, YAN and ANVERS records, Geochemical Zones (GZ) in the ARD and YAN records, and Guano phase GP-1 to GP-5 boundaries in the ARD record. Downcore cluster-zonation was undertaken on ecologically grouped diatom percentage data and square-root transformed geochemical data using the R packages vegan and rioja (see Supplementary Methods for software references). Downcore trends in diatom and geochemical data sets using Principal Components Analysis (PCA) were undertaken using square-root transformed percentage XRF data and m-XRF (Total Scatter Normalised (TSN)) percentage geochemical data and percentage diatom count data. Gy11% ¼ 0:11 DPg ¼ 847 300 g a 1p 1 ð8Þ ð8Þ Gy11% ¼ 0:11 DPg ¼ 847 300 g a 1p However, small lakes with restricted inﬂow and/or low rates of catchment erosion or slow sedimentation rates (that is, most typically high latitude, frozen lakes, for example, Yanou Lake during the late Holocene) are better represented by an exponential sediment delivery model, GDST (ref. 81), which, for Ardley Lake, produces GDST ¼ 0:96% as follows: GDST ¼ 0:36 A 0:2 max ¼ 0:36 7; 274 0:2 ¼ 0:96%: ð9Þ ð9Þ This allowed us to estimate the number of penguins (P) present in the catchment at time (t) for three guano-yield scenarios, Gy100%;11%;0:96%: This allowed us to estimate the number of penguins (P) present in the catchment at time (t) for three guano-yield scenarios, Gy100%;11%;0:96%: For climate and sea-ice comparisons, the Yanou Lake reconstructed GDGT temperature (YAN-GDGT) data (grey plot, with RMSE errors shaded grey in Supplementary Fig. 16a) and the Maxwell Bay measured TOC data (sea-ice proxy record)52 (grey plot in Fig. 4c) were smoothed using polynomial negative exponential regression analysis. Results were similar to smoothing by LOESS ﬁrst- order polynomial regression with tri-cube weighting (not shown). Reconstructed GDGT temperatures reﬂect MSAT (December, January, February or DJF) in the shallow and well-mixed open lake water environment of Yanou Lake76,77. ¼ Pðnumber of penguinsÞ To further validate our ﬁndings, we also calculated the catchment area required by the reconstructed colony population using published gentoo penguin density values40 and calculated mixed-species density values, partitioned according to published modern-day Ardley Island gentoo-Ade´lie-Chinstrap species compositions41 and the corresponding species density values40 (Supplementary Fig. 19b, Supplementary Table 9). After smoothing, all climate (YAN-GDGT, JRI29 and PD48); and sea-ice (Anvers Shelf and Maxwell Bay52) data sets were resampled at 100-year intervals (see Supplementary Methods). We then used 46–0 ka upper bound on the weighted mean (95% conﬁdence level) temperature, temperature anomaly, diatom open water ratio and smoothed TOC- and diatom-based sea-ice proxy data (Fig. 4) to deﬁne ‘warm’ and ‘sea-ice free’ (open-water) periods. We chose the 6–0 ka period and values greater than the upper bound on the weighted mean at 95% conﬁdence level of the weighted mean as this provides the best longer-term assessment of the mean temperature and variability that penguin colonies on Ardley Island would have encountered. It also corresponds to the time period covered by the terrestrial Yanou Lake GDGT-MSAT record. Using the Pre-industrial era (1,000–250 cal a BP) and its upper bound on the weighted mean value (95% conﬁdence) produced similar timings for ‘warm’ and ‘open-water’ intervals. y pp y g pp y The ‘most-likely’ minimum guano-delivery (yield) scenario assumes that 11% of the guano produced by penguins in the Ardley Lake catchment was eroded into Ardley Lake. This scenario produced a peak guano phase GP-4 colony population of 1,024±574 penguins, which occupied an area of 1,679–3,303 m2 out of the 66,249 m2 Ardley Lake catchment area (based on partitioned mixed species minimum density–gentoo penguin maximum density values40). Meanwhile, the 0.96% guano-yield scenario produced a maximum peak GP-4 colony population of 11,723±5,104, which corresponds to an occupied area of 20,686–37,815 m2 (Supplementary Fig. 19). Therefore, the c. 66,250 m2 Ardley Lake catchment area is easily large enough to accommodate all modelled population scenarios shown in Fig. 5. Assuming a hyperboloid basin shape for Ardley Lake would result in an average 33±25% reduction in the reconstructed population across all guano delivery scenarios. To compare variations in cross-Peninsula climate during guano and non-guano phases, we undertook normality tests and produced a set of descriptive statistics for the whole Ardley Fo.sed. ¼ Pðnumber of penguinsÞ ¼ Pðnumber of penguinsÞ Since the catchment colony would have produced and/or deposited guano outside the catchment area (for example, on foraging trips), and not all guano deposited in catchment areas is currently eroded into lake due to various factors, for example, utilization by vegetation, we consider the Gy–100% to be very unlikely. Therefore, we consider the Gy–11% and IGAo30ellip tð Þ to best represent a possible ‘most likely’ minimum population scenario as follows: Since the catchment colony would have produced and/or deposited guano outside the catchment area (for example, on foraging trips), and not all guano deposited in catchment areas is currently eroded into lake due to various factors, for example, utilization by vegetation, we consider the Gy–100% to be very unlikely. Therefore, we consider the Gy–11% and IGAo30ellip tð Þ to best represent a possible ‘most likely’ minimum population scenario as follows: MINPAo30ellipðtÞ ¼ IGAo30ellipðtÞ Gy11% Units : g a 1 g a 1p 1 ð11Þ ¼ Pðnumber of penguinsÞ ð11Þ ¼ Pðnumber of penguinsÞ ¼ Pðnumber of penguinsÞ ¼ Pðnumber of penguinsÞ As a reality check, the Gy-11% and IGAo30ellipðtÞ scenario recreated the near- absence of a penguin colony (o10 penguins) in the modern day catchment. Conversely, we consider that the Gy-0.96% and IGAo30ellipðtÞ scenario represents the ‘most likely’ maximum population scenario: As a reality check, the Gy-11% and IGAo30ellipðtÞ scenario recreated the near- absence of a penguin colony (o10 penguins) in the modern day catchment. As a reality check, the Gy-11% and IGAo30ellipðtÞ scenario recreated the near- absence of a penguin colony (o10 penguins) in the modern day catchment. y y % pð Þ absence of a penguin colony (o10 penguins) in the modern day catchment. Conversely, we consider that the Gy-0.96% and IGAo30ellipðtÞ scenario represents the ‘most likely’ maximum population scenario: Conversely, we consider that the Gy-0.96% and IGAo30ellipðtÞ scenario represents the ‘most likely’ maximum population scenario: MAXPAo30ellipðtÞ ¼ IGAo30ellipðtÞ Gy0:96% Units : g a 1 g a 1p 1 ð12Þ ¼ Pðnumber of penguinsÞ ð12Þ ¼ Pðnumber of penguinsÞ ð12Þ These two end-member scenarios are shown in Fig. 5, but since sedimentation rates in Ardley Lake have varied signiﬁcantly through time, it is possible that, when erosion rates are high, for example, between c. 5.5 and 4.5 cal ka BP, the Ardley Island colony population would be better estimated by a guano-yield scenario somewhere between 11 and 100%. ARTICLE ARTICLE NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 For the YAN record, whole YAN-GDGT data set 5th and 95th percentile outliers (o 0.33 C and 48.61 C) 11.34 C and 13.84 C at 1,164 cal a BP and 3,135 cal a BP, respectively, were excluded by smoothing analysis (Fig. 4a,c, Supplementary Fig. 17a). These values were also greater than the 10.3 C maximum limit of the surface sediment-MSAT Antarctic and sub-Antarctic GDGT-MSAT calibration (ANT-GDGT) data set77. All reconstructed GDGT temperatures were greater than the 2.2 C minimum limit of the ANT-GDGT calibration data set. The YAN- GDGT o10 C data set was used in 6–0 ka weighted mean temperature anomaly calculations, statistical analyses and hypothesis testing. The mean±1s 6–0 ka reconstructed YAN-GDGT o10 C data set weighted mean MSAT value of þ 2.39±2.67 C [95% conﬁdence interval: 1.52–3.25 C] (Bootstrapping, n ¼ 10,000, þ 2.38±2.61 C [1.52–3.25 C]; 5th/95th percentile ¼ 0.51/ þ 7.96 C; weighted by Antarctic GDGT calibration data set RMSE value of 1.45 C; Supplementary Table 7) encompasses the modern-day (1968–2015 CE) observational mean summer (DJF) temperature from Bellingshausen Research Station on Fildes Peninsula of þ 1.09±0.56 C (2s 95% measured range ¼ 0.03 to 2.21 C; n ¼ 48, 1968–2015 CE, where year refers to December) (Marshall, pers. comm.; SCAR-READER database: http://www.antarctica.ac.uk/met/READER/). On a regional to global-scale modern-day reconstructed lake sediment GDGT- temperatures broadly reﬂect changes in air temperature77. At the local, small lake scale, water temperature might decouple from observed MSAT due to factors such as the thickness and length of seasonal lake ice-cover and the volume of meltwater input, both of which suppress lake water temperatures. Conversely, since water bodies store excess heat generated during extended ice-free seasons, small and/or shallow lakes (o10 m deep) reach temperatures of 6 C or more (for example, Sombre Lake, Signy Island)82. As the focus of this paper is penguin colony reconstruction, we investigate these aspects further in a forthcoming paper. Gy100% ¼ 7; 716 2; 728 g a 1p 1 Gy11% ¼ 847 300 g a 1p 1 Gy0:96% ¼ 74 26 g a 1p 1 as follows as follows PAo30ellipðtÞ ¼ IGAo30ellipðtÞ Gy100%;11%;0:96% Units : g a 1 g a 1p 1 ð10Þ ¼ Pðnumber of penguinsÞ ð10Þ ¼ Pðnumber of penguinsÞ ð1 ARTICLE values GD ¼ Gy100% Ge ¼ Alake Acatch ¼ 7; 274 66; 249 m2 m2 ¼ 1 : 9 ¼ 11% ð7Þ ð7Þ where GD is the ratio, expressed as a percentage, of the total guano produced in the catchment (Gy-100%) and the total guano-eroded from the catchment (Ge) into the lake, which, here we approximate to the lake area: lake catchment ratio, Alake=Acatch. Alake=Acatch. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 12 ARTICLE 22) and is present in the JRI ice core29. Neither the 1976 or 1970 eruptions formed visible ash layers near the top of the ARD core, probably because only up to 1 mm of ash was deposited on Fildes Peninsula21. Therefore, we broadly equated major VEI ¼ 3 eruptions from Deception to visible tephra deposits o1 cm thick in the YAN and ARD records, and airfall ash deposits 41 cm thick to VEI43 eruptions. Since the post-deglaciation (after c. 8–7.5 cal ka BP) lithogenic deposition style in YAN and ARD is predominantly ﬁne-grained silt-clay, the sand dry mass accumulation rate (DMAR) provided a useful proxy for eruption size and the volume of tephra deposited onto Ardley Island and King George Island during VEI 3 i (Fi 4 b S l Fi 12 20 21) hil l d C /Ti 18. Li, C. et al. Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment. Gigascience 3, 27 (2014). g 19. Levy, H. et al. Population structure and phylogeography of the Gentoo Penguin (Pygoscelis papua) across the Scotia Arc. Ecol. Evol. 6, 1834–1853 (2016). 20. Bartolini, S., Geyer, A., Martı´, J., Pedrazzi, D. & Aguirre-Dı´az, G. 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All data produced by this study and associated IMCOAST/IMCONET projects are available from the corresponding author (sjro@bas.ac.uk) and the NERC Polar Data Centre (BAS) (https://www.bas.ac.uk/data/uk-pdc/) and Pangaea Data Publisher website (AWI) (https://www.pangaea.de/). Antarctic location maps and spatial data in Fig. 1 are the LIMA (Landsat Image Mosaic of Antarctica) downloaded from https://lima.usgs.gov/index.php and the SCAR Antarctic Digital Database http://www.add.scar.org/, used under a Creative Commons license. The basemap in Fig. References Nature 535, 411–415 (2016). 37. Meredith, M. P. & King, J. C. Rapid climate change in the ocean west of the Antarctic Peninsula during the second half of the 20th century. Geophys. Res. Lett. 32, 1–5 (2005). 8. Cook, A. J. et al. Ocean forcing of glacier retreat in the western Antarctic Peninsula. Science 353, 283–286 (2016). 38. Trivelpiece, W. Z. et al. 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Differential advancement of breeding phenology in response to climate may alter staggered breeding among sympatric pygoscelid penguins. Mar. Ecol. Prog. Ser. 454, 135–145 (2012). 35. Cimino, M. A., Lynch, H. J., Saba, V. S. & Oliver, M. J. Projected asymmetric response of Ade´lie penguins to Antarctic climate change. Sci. Rep. 6, 28785 (2016). 6. Southwell, C. et al. Spatially extensive standardized surveys reveal widespread, multi-decadal increase in East Antarctic Ade´lie penguin populations. PLoS ONE 10, e0139877 (2015). 36. Sun, L. et al. Vertebrate records in polar sediments: biological responses to past climate change and human activities. Earth-Science Rev. 126, 147–155 (2013). 7. Turner, J. et al. Absence of 21st century warming on Antarctic Peninsula consistent with natural variability. ARTICLE 1a and Supplementary Figs 20,23 is the ESRI Ocean Basemap (http://goto.arcgisonline.com/maps/Ocean_Basemap) used under a BAS-NERC software license. Satellite images and image derivatives in Figs 1c,d and 6 and Supplementary Figs 2,22,23 are copyright of DigitalGlobe, Inc. All Rights Reserved (Catalogue numbers: 1030010020C0C900 and 11MAR04115702), reproduced at low resolution in a static format under a BAS-NERC educational license, and are not included in the Creative Commons license for the article. 24. Smellie, J. L. Lithostratigraphy and volcanic evolution of Deception Island, South Shetland Islands. Antarct. Sci. 13, 188–209 (2001). 25. Newhall, C. G. & Self, S. The volcanic explosivity index (VEI) an estimate of l i it d f hi t i l l i J G ph R 87 1231 (1982) 25. Newhall, C. G. & Self, S. The volcanic explosivity index (VEI) an estimate of explosive magnitude for historical volcanism. J. Geophys. Res. 87, 1231 (1982). 26. Ben-Zvi, T. et al. The P-wave velocity structure of Deception Island, Antarctica, from two-dimensional seismic tomography. J. Volcanol. Geotherm. Res. 180, 67–80 (2009). 27. Toro, M. et al. Chronostratigraphy of the sedimentary record of Limnopolar Lake, Byers Peninsula, Livingston Island, Antarctica. Antarct. Sci. 25, 198–212 (2013). 28. Bjo¨rck, S., Håkansson, H., Zale, R., Karlen, W. & Jo´nsson, B. L. A late Holocene lake sediment sequence from Livingston Island, South Shetland Islands, with palaeoclimatic implications. Antarct. Sci. 3, 61–72 (1991). 29. Mulvaney, R. et al. Recent Antarctic Peninsula warming relative to Holocene climate and ice-shelf history. Nature 489, 141–144 (2012). ¼ Pðnumber of penguinsÞ data set (weighted by 1s error), the YAN-GDGT o10 C temperature data set, the JRI temperature anomaly data set29, and the PD 0–200 m sea-surface temperature (PD-SST) record48 (weighted by published errors, where available). Using a ¼ 5% and 10% signiﬁcance levels for three hypothesis test scenarios A–C shown in Supplementary Table 8, we ﬁrst performed Fisher’s F-test Variance Ratio analysis. Since some data sets were not normally distributed, we then used the Mann–Whitney U-statistic to test the hypothesis that temperature and temperature anomaly distributions in the YAN-GDGT o10 C, JRI, PD-SST records were statistically different during the ﬁve guano-inﬂuenced phases (Scenario 1A in Supplementary Table 8) compared to the eight non-guano phases (Scenario 2A). We then repeated this process comparing guano phases of the mid- Holocene (8.2–4.2 ka; 1B) to those of the late Holocene (4.2–0 ka; 2B), and for late Holocene guano phases (1C) versus non-guano phases (2C). We also undertook similar hypothesis testing using the Maxwell Bay sea-ice and Anvers Shelf open- water data sets to determine whether sea-ice conditions within guano and non- guano were statistically different in the three scenarios (A–C) described above. Results are summarized in Supplementary Table 8. It is important to note that the apparent increase in the amount of guano deposited into Ardley Lake over the last 500 years is less than the signiﬁcant 10% Fo.sed. level we used to determine colony presence elsewhere in its Holocene record, and only equates to a total of 65±48 penguins (using the 11% guano-delivery scenario). This also illustrates why the Ardley Lake guano phase colony was too small after c. 2,500 years for us to determine whether volcanic activity had a signiﬁcant impact. Correlation analysis of geochemical data was undertaken using R 2.15.2 (R Foundation for Statistical Computing). Hierarchical R-mode cluster analyses were conducted with the R package ‘Pvclust’ (version 1.2-2) using average linkage and a correlation-based dissimilarity matrix (see Supplementary Methods for software references). Based on multi-scale bootstrap resampling (number of bootstraps: 10,000), approximately unbiased P values were further calculated to 13 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Apart from one recent minor eruption, all visible (that is, 42 mm thick) Holocene-age tephra deposits in lake, marine and ice core records of the northern Antarctic Peninsula (N-AP), SSI and Scotia Sea have been linked to VEI ¼ 3 or VEI43 eruptions from Deception Island20–23,27–29,58,63,83,84 (Supplementary Note 9, Supplementary Figs 20, 21, Supplementary Tables 3, 10). Tephra from even relatively small 1967 and 1970 CE Deception Island eruptions (VEI ¼ 3) reached more than 150 km (ref. 22) and is present in the JRI ice core29. Neither the 1976 or 1970 eruptions formed visible ash layers near the top of the ARD core, probably because only up to 1 mm of ash was deposited on Fildes Peninsula21. Therefore, we broadly equated major VEI ¼ 3 eruptions from Deception to visible tephra deposits o1 cm thick in the YAN and ARD records, and airfall ash deposits 41 cm thick to VEI43 eruptions. Since the post-deglaciation (after c. 8–7.5 cal ka BP) lithogenic deposition style in YAN and ARD is predominantly ﬁne-grained silt-clay, the sand dry mass accumulation rate (DMAR) provided a useful proxy for eruption size and the volume of tephra deposited onto Ardley Island and King George Island during VEI43 eruptions (Fig. 4a,b, Supplementary Figs 12,20,21), while elevated Ca/Ti ratios and PCA2 values from combined 2 mm and 200 mm m-XRF scanning data and the reliable single-species aquatic moss age-depth model provided precise stratigraphic constraints on the positions (and ages) of tephra deposits in the YAN record (Supplementary Note 9, Supplementary Figs 9–11,16, Supplementary Table 3). Apart from one recent minor eruption, all visible (that is, 42 mm thick) Holocene-age tephra deposits in lake, marine and ice core records of the northern 16. Emslie, S. D., Polito, M. J., Brasso, R., Patterson, W. P. & Sun, L. Ornithogenic soils and the paleoecology of pygoscelid penguins in Antarctica. Quat. Int. 352, 4–15 (2014). g p p Antarctic Peninsula (N-AP), SSI and Scotia Sea have been linked to VEI ¼ 3 or VEI43 eruptions from Deception Island20–23,27–29,58,63,83,84 (Supplementary Note 17. Clucas, G. V. et al. A reversal of fortunes: climate change ‘winners’ and ‘losers’ in Antarctic Peninsula penguins. Sci. Rep. 4, 5024 (2014). 9, Supplementary Figs 20, 21, Supplementary Tables 3, 10). Tephra from even relatively small 1967 and 1970 CE Deception Island eruptions (VEI ¼ 3) reached more than 150 km (ref. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Acknowledgements This study forms part of the ESF-funded IMCOAST project, AP-6 led by S.J.R., and the EU project IMCONet (FP7404 IRSES, action no. 319718; coordinated by the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Germany) with additional funding from the Natural Environmental Research Council (NERC), and the German Research Foundation (DFG project no. BR 775/25-1) and logistic support from the NERC-British Antarctic Survey (BAS), HMS Endurance and 892 Naval Air Squadron, the Alfred Wegner Institute (AWI) and the Instituto Anta´rtico Argentino (IAA). We thank NERC for funding studentship NE/J500173/1 (BAS and Newcastle University) to L.C.F. (supervisors S.J.R., E.J.P., D.A.H., S.J.). We thank colleagues at the Chinese Great Wall Station and Russian Bellingshausen Bases on Fildes Peninsula for their generous hospitality, H. Biester and T. 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A 1300-year record of penguin populations at Ardley Island in the Antarctic, as deduced from the 14 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/ NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Additional information 71. Van Daele, M. et al. The 600 yr eruptive history of Villarrica Volcano (Chile) revealed by annually laminated lake sediments. Geol. Soc. Am. Bull. 126, 481–498 (2014). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 15 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications URE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications r The Author(s) 2017 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Competing interests: The authors declare no competing ﬁnancial interests. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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https://openalex.org/W2058213202	https://europepmc.org/articles/pmc3853476?pdf=render	English	null	Plasticity of primary microglia on micropatterned geometries and spontaneous long-distance migration in microfluidic channels	BMC neuroscience	2,013	cc-by	9,390	Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 * Correspondence: cinzia.volonte@cnr.it †Equal contributors 1Santa Lucia Foundation/CNR-Cellular Biology and Neurobiology Institute, Via del Fosso di Fiorano 65, 00143 Rome, Italy Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Abstract Background: Microglia possess an elevated grade of plasticity, undergoing several structural changes based on their location and state of activation. The first step towards the comprehension of microglia’s biology and functional responses to an extremely mutable extracellular milieu, consists in discriminating the morphological features acquired by cells maintained in vitro under diverse environmental conditions. Previous work described neither primary microglia grown on artificially patterned environments which impose physical cues and constraints, nor long distance migration of microglia in vitro. To this aim, the present work exploits artificial bio-mimetic microstructured substrates with pillar-shaped or line-grating geometries fabricated on poly(dimethylsiloxane) by soft lithography, in addition to microfluidic devices, and highlights some morphological/functional characteristics of microglia which were underestimated or unknown so far. Results: We report that primary microglia selectively adapt to diverse microstructured substrates modifying accordingly their morphological features and behavior. On micropatterned pillar-shaped geometries, microglia appear multipolar, extend several protrusions in all directions and form distinct pseudopodia. On both micropatterned line-grating geometries and microfluidic channels, microglia extend the cytoplasm from a roundish to a stretched, flattened morphology and assume a filopodia-bearing bipolar structure. Finally, we show that in the absence of any applied chemical gradient, primary microglia spontaneously moves through microfluidic channels for a distance of up to 500 μm in approximately 12 hours, with an average speed of 0.66 μm/min. Conclusions: We demonstrate an elevated grade of microglia plasticity in response to a mutable extracellular environment, thus making these cells an appealing population to be further exploited for lab on chip technologies. The development of microglia-based microstructured substrates opens the road to novel hybrid platforms for testing drugs for neuroinflammatory diseases. Keywords: Confocal analysis, Long distance migration, Microglia plasticity, Microfabrication, Time-lapse microscopy Plasticity of primary microglia on micropatterned geometries and spontaneous long-distance migration in microfluidic channels Amadio et al. Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Plasticity of primary microglia on micropatterned geometries and spontaneous long-distance migration in microfluidic channels Susanna Amadio1†, Adele De Ninno2,3†, Cinzia Montilli1, Luca Businaro2, Annamaria Gerardino2 and Cinzia Volonté1* © 2013 Amadio 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. © 2013 Amadio et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background plasticity, undergoing a variety of structural changes based on their location and particular state of activation. Unlike other cells in the brain, they are extremely versatile and dynamic [2]. They have the capacity to sense and adjust to the microenvironment, to migrate, proliferate and phago- cytose. During early development, microglia enter the brain using vessels and white matter tracts as guiding structure for migration, and then disperse throughout the CNS occupying a defined territory [3]. Late in develop- ment, they transform from an amoeboid morphology into a branched, ramified phenotype composed of long, Microglia are part of the immune system being the resi- dent macrophages of the brain and spinal cord, and were first discovered and defined by Pio Del Rio Hortega [1] as an independent cellular phenotype abundantly present in the central nervous system (CNS). Microglia are of mesodermal origin and possess an elevated grade of * Correspondence: cinzia.volonte@cnr.it †Equal contributors 1Santa Lucia Foundation/CNR-Cellular Biology and Neurobiology Institute, Via del Fosso di Fiorano 65, 00143 Rome, Italy Full list of author information is available at the end of the article Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 2 of 12 Figure 1 Micropatterned structures serving for primary microglia culturing. Microtopographic silicon masters are realized to replica-mold substrates on PDMS with pillar-shaped and line- grating geometries (width: 1500 nm, pitch: 3 μm, height: 550 nm). A. Scanning electron microscope (Zeiss EVO) images of PDMS pillar- shaped substrates. B. Scanning electron microscope images of Si master with line-grating structures. C. PDMS line-gratings at high magnification. constantly extending, shrinking and re-growing pro- cesses with small, fairly motionless cell bodies, known today as surveilling microglia. These shape-shifting cells constitute about 20% of the total glial cell population within the adult brain and act as the first and main form of active immune defense in the CNS, by frequently scavenging for pathogens, damaged or dead cells and misfolded proteins, but also trimming away weak or damaged synapses between neurons [4-6]. Indeed, after a pathological event, microglia respond to the environ- ment by undergoing profound transition in morphological appearance, motility and state of activation, and reacquir- ing an amoeboid shape similar to the one observed early in development. Background This high level of plasticity is required to fulfill the vast variety of immunological functions that microglia perform, as well as for maintaining homeostasis within the brain [2,7-12]. The first step towards the comprehension of microglia’s biology consists in discriminating if different morpho- logical features can be acquired in vitro when microglia are cultured on diverse surface topography that are mi- metic of an in vivo resembling three-dimensional (3D) en- vironment. To this aim, we employed microstructured pillar-shaped and line-grating geometries produced on poly(dimethylsiloxane) (PDMS) by standard soft lithog- raphy techniques, and two-layer microfabrication photo- lithography. While the use of artificial bio-mimetic microtextured substrates and microfluidic devices [13] was mostly exploited for cancer cells [14-16] and neu- rons [17,18], only few studies were performed so far on microglia [19,20]. Microglia spontaneously adjust to micropatterned structures Figure 1 Micropatterned structures serving for primary microglia culturing. Microtopographic silicon masters are realized to replica-mold substrates on PDMS with pillar-shaped and line- grating geometries (width: 1500 nm, pitch: 3 μm, height: 550 nm). A. Scanning electron microscope (Zeiss EVO) images of PDMS pillar- shaped substrates. B. Scanning electron microscope images of Si master with line-grating structures. C. PDMS line-gratings at high magnification. The first aim of our work is to identify some experimen- tal conditions that could distinguish the morphological states of microglia in vitro, since the variety of changes that microglia undergo in vivo depends on the hetero- geneity of the environment [21-23]. Due to the extreme variability of microglial immortalized cell lines (such as N9 or BV-2 cells), we mostly used primary microglia dis- sociated cultures from rat and mouse cerebral cortex [24] plated on micropatterned pillar-shaped (Figure 1A) and line grating (Figure 1B,C) substrates. Figure 2A illustrates primary microglia cultured on plastic dishes coated with fibronectin, a condition commonly adopted in vitro for motility studies. Double fluorescence con- focal analysis performed with phalloidin (a marker for filamentous actin, in green) and P2Y12 receptor anti- serum (a marker for microglia, in red) [25] shows the heterogeneous morphological features of microglia in culture, likely representing different functional states (surveillance, scavenge, migration, phagocytosis, antigen presentation, cytotoxicity). Indeed, we simultaneously observe roundish cell bodies with single, long processes enriched by short and tiny branches (a); asymmetrical “hairy” cells with miniature processes (b); elongated “rod-like” cell bodies with no or few branches (c); amoeboid cells (d); cells crowned by lamellipodia (e), or possessing several filopodia (f) or fluffy fan-like cytoplas- mic protrusions (g). When instead cultured on biocompat- ible microstructured PDMS substrates, microglia undergo a remarkable homogeneous metamorphosis. On pillar- shaped geometries (Figures 1A and 2B, 2C), the majority of cells appears multipolar, with protuberances spreading Page 3 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Figure 2 Microglia adjust to micropatterned structures. A. Primary mouse microglia are cultured on plastic dishes and subjected to immunofluorescence and confocal analysis with phalloidin (green) plus P2Y12 receptors antiserum (red) and Hoechst (blue), scale bar = 50 μm. B-C. Primary microglia are cultured on pillar microstructures subjected to fluorescence and confocal analysis with phalloidin (green) plus Hoechst (blue), scale bar = 20 μm (B), or immunofluorescence with paxillin (red), scale bar = 10 μm (C). D. Microglia spontaneously adjust to micropatterned structures Primary mouse microglia are cultured on line- grating geometries and subjected to fluorescence and confocal analysis with phalloidin (green) plus Hoechst (blue), scale bar = 20 μm. Figure 2 Microglia adjust to micropatterned structures. A. Primary mouse microglia are cultured on plastic dishes and subjected to immunofluorescence and confocal analysis with phalloidin (green) plus P2Y12 receptors antiserum (red) and Hoechst (blue), scale bar = 50 μm. B-C. Primary microglia are cultured on pillar microstructures subjected to fluorescence and confocal analysis with phalloidin (green) plus Hoechst (blue), scale bar = 20 μm (B), or immunofluorescence with paxillin (red), scale bar = 10 μm (C). D. Primary mouse microglia are cultured on line- grating geometries and subjected to fluorescence and confocal analysis with phalloidin (green) plus Hoechst (blue), scale bar = 20 μm. line-grating geometries, we find that M is 62.41 ± 21.98 μm or 116.20 ± 56.54 μm, m is 23.36 ± 7.84 μm or 11.36 ± 3.18 μm, AR is 2.83 ± 1.26 or 11.01 ± 6.86 (Figure 3C). out from roundish or fairly oblongated cell bodies, forming button-shaped lamellipodia or pseudopodia. These protrusions furthermore highlight the subjacent pillar structures (phalloidin staining, panel B or paxillin, panel C), and are likely to reflect points of membrane adhesion to the underlying substrate. On line-grating geometries (Figures 1B,C and 2D), microglia become longitudinally flattened on the substrate and show a filopodia-bearing elongated bipolar structure [26]. Morphological transition of microglia in microfluidic devices We then asked if the elongated bipolar structure of microglia on line-grating geometries might convert into propensity for instance to motility, when cells are cul- tured on microfluidic devices (Figure 4). By immuno- fluorescence and confocal analysis, we observe that primary cortical microglia cultures (Figure 5, left panel) are very different from N9 microglia cells (Figure 5, right panel) [29,30], in terms of shape and morphological ad- justment to the microchannels. When residing in the culturing chamber of the microfluidic device (labeled as cc in Figures 4 and 5), between the proper microfluidic channels (labeled as mc in Figures 4 and 5) and the reservoir (round grey area in Figure 4), the majority of pri- mary microglia (Figure 5, left panel) appears heteroge- neous, with a large diameter (20–50 μm) but few and long processes (phalloidin in green; anti-P2Y12 purinergic re- ceptor in red). Cells tend to aggregate when residing in the culturing chamber (cc) but, when present inside the microfluidic channels (mc), they extend the cytoplasm from a roundish to a stretched, elongated morphology, with a clear leading edge, similarly to what observed on line grating geometries (Figures 2D and 3B). Conversely, Primary microglia spontaneously migrate in microfluidic channels In order to prove if morphological transition affects base- line motility of primary microglia, we performed time- lapse recording on artificial microfluidic substrates with controlled characteristics (chemical composition, shape, dimensions, softness) [16]. Fibronectin-coated PDMS microfluidic microchannels with a width of 12–18 μm, a length of 500 μm (but not 3 mm), a reservoir chamber of 200 μl in volume, are found permissive to this aim (Figure 4). The presence of a fibronectin coating gradient potentially generated inside the microchannels and pos- sibly sustaining cell migration is moreover excluded by immunofluorescence confocal analysis in the presence of anti-fibronectin (Figure 6A,B). Under these conditions, we observe that several microglial cells are scattered in the culturing chamber (cc), with many cells apparently aligned in proximity to the microchannels (Figure 6C, microglial marker IBA1 [31] in red). Moreover, we detect microglia also engaged inside the microchannels (mc), with some cells finally moving out from the microchannels, after hav- ing completed the 500 μm full length (white dotted arrow). When time-lapse is captured for 20 h every 30 mi- nutes (Figure 7 and Additional file 1), we observe that at T = 0 min, many round-shaped microglial cells are posi- tioned in proximity to the microchannel openings, at the boundary between microchannels and culturing chamber. At T = 30 min, several microglial processes are already surveilling the environment and entering into the micro- channels (arrow), with a few cells already engaged inside the channels (arrowhead). At T = 3.5 h, several cells are committed and already moving inside the channels (arrowhead). At T = 4 h, some cells move forward (white, pink, green, red, yellow arrowhead), some stay still (black arrowhead) or move backward in the microchannels (pur- ple arrowhead at T = 3.5 h). At T = 5.5 h, two cells (orange and light blue arrowhead) are entering in microchannels already occupied by other cells (respectively white and blue). Between T = 5.5 and T = 11.5 h, some cells change direction and move backward (pink, yellow arrowhead), some stop moving after a displacement of about 120– 150 μm (green, red arrowhead) or about 250 μm (blue Figure 3 Morphometric analysis of microglia on round-shaped N9 cells have an average diameter of about 20 μm, possess numerous very short processes and have the tendency to disorderly coalesce when residing in the culturing chamber (Figure 5, right panel, phalloidin in green, cc). Morphometric analysis of microglia on micropatterned structures These morphologic observations are confirmed by quanti- tative analysis performed importing the fluorescence and confocal microscopy images into Matlab. We adopted cir- cularity ratio (CR) and axis ratio (AR) shape descriptors. CR defines the ratio between the area of the shape of inter- est and the area of a circle having the same perimeter [27]. This parameter represents how the shape of interest differs from a circle, and it is expressed as CR = 4πAP-2, where “A” is the cell area and “P” the cell perimeter length. AR defines the ratio between the major axis (M) and the minor axis (m) of the cell’s fitted ellipse [28], and represents a measure of how the shape of interest is “elongated”. In de- tail, we observe that on pillar (Figure 3A,C) or line-grating (Figure 3B,C) geometries, the average M, m and AR of microglia are different, remaining approximately constant the average P, A and CR values. Respectively on pillar or Page 4 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 however maintain quite constant their average A, AR and CR (Table 1). These parameters are instead very different in primary microglia present inside the microchannels, or in the culturing chamber. They are A: 523 ± 52 μm2 versus 1807 ± 545 μm2; AR: 20 ± 9 versus 1.36 ± 0.19; CR: 0.08 ± 0.03 versus 0.36 ± 0.12. Moreover, we also observe that both morphological appearance and average values of A, AR and CR are very different in polarized primary micro- glia versus not-polarized N9 cells (Table 1), especially in the microchannels, thus discouraging the use of these last for motility studies. round-shaped N9 cells have an average diameter of about 20 μm, possess numerous very short processes and have Figure 3 Morphometric analysis of microglia on micropatterned structures. Morphometric analysis of primary microglia cultured on pillar microstructures (A) and line-grating geometries (B) is performed using a customized Matlab code (C). Values are expressed as mean ± SD, with n = 14 cells on pillars or line-grating structures. A significative difference is obtained for all parameters by Mann–Whitney test (p < 0.0003). “M” indicates major axis and “m” minor axis of cell’s fitted ellipse (panels A, B), “A” cell area, “P” cell perimeter, “AR” aspect ratio, “CR” circularity ratio. Primary microglia spontaneously migrate in microfluidic channels When engaged inside the microfluidic channels (mc), they acquire a quadrangle shape, are strictly aligned to each other by cell contacts, as suggested by the pres- ence of stronger phalloidin fluorescent signals at the cell- to-cell side. In the two different compartments, N9 cells Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 5 of 12 Figure 4 Microfluidic structure adopted for primary microglia culturing. Schematic representation of the microfluidic device used for the microglia motility experiments. Reservoirs, culturing chambers (cc) and microchannels (mc) areas are highlighted. Figure 4 Microfluidic structure adopted for primary microglia culturing. Schematic representation of the microfluidic device used for the microglia motility experiments. Reservoirs, culturing chambers (cc) and microchannels (mc) areas are highlighted. arrowhead). At T = 11.5 h, we also observe a cell complet- ing the 500 μm full length and moving out from the other side (white arrowhead) (Figure 7 and Additional file 1). Moreover, we detect: cells branching and alternating their processes in and out from the same microfluidic channel, or in more than one channel; cell bodies oscillating be- tween two adjacent channels; two cells proceeding simul- taneously inside a single channel; cells inverting direction and moving out from the channels; cells overtaking each other inside the channels (Additional file 1). During the time-lapse, the cells always appear healthy and possess a roundish or elongated shape respectively in the culturing chamber and the microchannels. percentage of total cells engaged in the microchannels is about 50% of those residing in the culturing chamber; c) the maximal distance covered by a single cell in the microchannel is 500 μm in 11.5 h; d) the average accumu- lated distance of microglia somata over 20 h recording is 409.2 ± 169.6 μm in the microchannels, with respect to 125.6 ± 79.5 μm in the culturing chamber (Figure 8A); e) the highest velocity is 0.66 μm/min; f) the mean velocity of microglia somata inside the microchannels is 0.350 ± 0.145 μm/min, as compared to 0.107 ± 0.068 μm/min in the culturing chamber (Figure 8B). Moreover, also microglia processes (defined as cytoplasm protrusion with a length equal to at least one cell body diameter) are found extremely motile, undergoing extensions and retractions when moving inside the microchannels. The maximal length change of individual processes inside the microchannels is 70–80 μm. Primary microglia spontaneously migrate in microfluidic channels To quantify the morpho- logical changes, we analyzed cells present in the different compartments of the microfluidic device (Figure 8C). In- side the microchannels (n = 79 cells), we observe a 50% decrease in the average CR (0.25 ± 0.12), with respect to Discussion Microglia consist of at least two subpopulations that co- exist in the adult CNS and derive from different sources: one that originates from bone marrow-derived cells and migrates to the CNS during embryonic development for colonization of the nervous system parenchyma, the sec- ond that develops from myeloid progenitor cells and en- ters the brain after birth [32], becoming fundamental for microglial maintenance of the CNS homeostasis [6,33]. Since adult microglia can play a twofold role, either ampli- fying the effects of inflammation and mediating cell degeneration, or protecting the nervous system from pathological insults [6], efficient chemotaxis can acquire either neuroprotective or inflammatory and detrimental roles. Whereas we are currently capable of distinguishing amoeboid-phagocytic from branched-surveilling microglia [3,5], we are unaware of how these morphological shapes can denote a neuroprotective or neurotoxic role. In other words, it is still an open matter how the vast morpho- logical heterogeneity existing within the mixed microglia population might relate to functional diversification. Cell behavior strictly depends on the stiffness and shape of the microenvironment, and the response to surface top- ography is a very critical determinant of cell morphogen- esis. Microglia respond to micro- nano-structured pits, protrusions and grooves with altered morphology, adhe- sion, and directional growth [19]. In our work, we estab- lish that primary microglia retain in vitro the intrinsic competence of modifying their structure in response to contact guidance cues. When grown on pillar-shaped substrates, microglia acquire a center-stage multi-polar morphology and develop abundant button-like pseudopo- dia emerging from the cell body with a nearly radial orien- tation. This situation might very well depict the in vivo condition of microglia not committed to migrate, but ex- ploring the environment with short forward, backward and sideways steps, with the final purpose to orient their migration. When placed on a line-grating substrate in the presence of parallel and symmetrical grooves, microglia elicit a longitudinally flattened appearance with elongated bipolar shape. This in vitro condition might favor a polar- ized extension of filopodia at the leading edge of the cell, in preparation of a forward translocation of the cell body, with retraction of the rear of the cell [26]. The adjustment Figure 6 Migration of primary microglia in microfluidic devices occurs on homogeneous fibronectin coating. A. A homogeneous Figure 6 Migration of primary microglia in microfluidic devices occurs on homogeneous fibronectin coating. A. A homogeneous fibronectin coating is formed inside the microchannels. Morphometric analysis of primary microglia migrating in microfluidic channels Quantitative analysis of individual somata performed dur- ing the time-lapse confirms the highly dynamic nature of microglia. By analyzing image stacks and time series with Image J software, we observe that: a) only 2 ~ 3 cells are found simultaneously inside a single microchannel; b) the Figure 5 Morphological transition of microglia in microfluidic devices. Primary rat microglia (left panel) and N9 microglia cell line (right panel) are maintained in culture on microfluidic devices for 24 hours and subjected to immunofluorescence and confocal analysis with phalloidin (green) plus P2Y12 receptors antiserum (red) (left panel, scale bar = 50 μm) or fluorescence with phalloidin (right panel, scale bar = 20 μm). mc indicates the microchannels areas and cc shows the culturing chambers, as schematically depicted in Figure 4. Figure 5 Morphological transition of microglia in microfluidic devices. Primary rat microglia (left panel) and N9 microglia cell line (right panel) are maintained in culture on microfluidic devices for 24 hours and subjected to immunofluorescence and confocal analysis with phalloidin (green) plus P2Y12 receptors antiserum (red) (left panel, scale bar = 50 μm) or fluorescence with phalloidin (right panel, scale bar = 20 μm). mc indicates the microchannels areas and cc shows the culturing chambers, as schematically depicted in Figure 4. Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 6 of 12 Table 1 Morphometric analysis of microglia in microfluidic devices Primary microglia microchannels Primary microglia microchambers N9 cells microchannels N9 cells microchambers A (μm2) 523 ± 52 1807 ± 545 487 ± 176 680 ± 141 AR 20 ± 9 1.36 ± 0.19 1.56 ± 0.38 1.51 ± 0.34 CR 0.08 ± 0.03 0.36 ± 0.12 0.25 ± 0.14 0.39 ± 0.2 cells (n = 120) in the culturing chamber (CR = 0.48 ± 0.18), while the average AR increases about twofold. Morphometric analysis of primary microglia migrating in microfluidic channels Table 1 Morphometric analysis of microglia in microfluidic devices Primary microglia microchannels Primary microglia microchambers N9 cells microchannels N9 cells microchambers A (μm2) 523 ± 52 1807 ± 545 487 ± 176 680 ± 141 AR 20 ± 9 1.36 ± 0.19 1.56 ± 0.38 1.51 ± 0.34 CR 0.08 ± 0.03 0.36 ± 0.12 0.25 ± 0.14 0.39 ± 0.2 Table 1 Morphometric analysis of microglia in microfluidic devices Table 1 Morphometric analysis of microglia in microfluidic devices Primary microglia microchannels Primary microglia microchambe y g y g A (μm2) 523 ± 52 1807 ± 545 AR 20 ± 9 1.36 ± 0.19 CR 0.08 ± 0.03 0.36 ± 0.12 Figure 6 Migration of primary microglia in microfluidic devices occurs on homogeneous fibronectin coating. A. A homogeneous fibronectin coating is formed inside the microchannels. B. The profile of fluorescence intensity acquired with Zen software of Zeiss LSM 700 microscope provides values ranging from 3 to 55 fluorescence intensity arbitrary units. C. Microglial cells are cultured in microfluidic devices and subjected to immunofluorescence for IBA1 (red) and staining with Hoechst (blue). The total migration path is: length 500 μm (white dotted arrow), width 12 μm, height 10 μm, and the scale bar is 50 μm. cells (n = 120) in the culturing chamber (CR = 0.48 ± 0.18), while the average AR increases about twofold. Discussion B. The profile of fluorescence intensity acquired with Zen software of Zeiss LSM 700 microscope provides values ranging from 3 to 55 fluorescence intensity arbitrary units. C. Microglial cells are cultured in microfluidic devices and subjected to immunofluorescence for IBA1 (red) and staining with Hoechst (blue). The total migration path is: length 500 μm (white dotted arrow), width 12 μm, height 10 μm, and the scale bar is 50 μm. Page 7 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Amadio et al. BMC Neuroscience 2013, 14:121 Figure 7 Primary microglia spontaneously migrate in microfluidic channels. After plating primary microglia in microfluidic devices for 30 min, time-lapse recording is performed every 30 min for 20 h. Arrows represent microglial processes and arrowheads indicate microglial cells. The different colors point to different forward- or backward-displacement of the cells inside the microchannels. The total migration path is: length 500 μm (white dotted arrow), width 12 μm, height 10 μm. The scale bar is 50 μm. Figure 7 Primary microglia spontaneously migrate in microfluidic channels. After plating primary microglia in microfluidic devices for 30 min, time-lapse recording is performed every 30 min for 20 h. Arrows represent microglial processes and arrowheads indicate microglial cells. The different colors point to different forward- or backward-displacement of the cells inside the microchannels. The total migration path is: length 500 μm (white dotted arrow), width 12 μm, height 10 μm. The scale bar is 50 μm. adhesion, distribution of actin cytoskeleton and microtu- bules, phagocytosis, antigen presenting power, beneficial or detrimental chemokine/cytokine expression and re- lease, will allow to determine the functional identity of multipolar versus bipolar microglia. of microglia to a line-grating geometry is thus highly suggestive of commitment to follow a straight path in vivo. Thus, surface topography can induce a syn- chronous and homogeneous metamorphosis of micro- glia in vitro, distinguishing the center-stage multipolar (on micropatterned pillars) from the elongated bipolar (on line-grating micropatterns) cells. With the use of specific markers and cytoskeleton-perturbing drugs, fu- ture work will aim to establish if these subpopulations can be correlated to: a) microglia generated from bone marrow rather than from myeloid progenitor cells; b) microglia colonizing the CNS during development, rather than maintaining CNS homeostasis in adulthood; c) more importantly, “beneficial” rather than “detrimental” micro- glia. Discussion brain, reaching up to several micrometers in length, or retracting until they completely disappear. This baseline dynamism of microglial processes is thus in sharp contrast to the stability of the microglial cell bodies and surround- ing neuronal processes [4]. contact formation, rather than the aptitude of primary cells to explore the environment with a direction- oriented polarization. In both cases, specific receptors and structural molecules are found enriched respect- ively at the cell-to-cell surface (N9 cells) or leading edges (primary microglia), as evinced by increased phal- loidin and P2Y12 receptor signals at these sites. In vitro studies on this subject have instead shown only transwell device infiltration directed by a chemical gradient. Lee and Chung [35] describe that ADP stimu- lates chemotaxis of immortalized BV2 microglia from the upper to the lower side of the transwell membrane. Karlstetter and co-authors [36] present evidence that curcumin inhibits basal and LPS-induced relocation of BV2 microglia from the upper to the lower transwell membrane surface [13]. However this passage across a physical barrier limits the possibility of investigating long distance migrations and, moreover, provides only indir- ect evaluation of kinetic parameters. Honda and collabo- rators [37] using the Dunn chemotaxis chambers report that primary rat microglia have weak motility in the ab- sence of ligands, but perform a mean displacement of about 50 μm in the presence of ATP or ADP. Nasu-Tada et al. [38] confirm that primary microglia are almost static in the absence of stimulants, but show chemotac- tic responsiveness to ADP with a maximal displacement of about 140 μm. Finally, Haynes and coauthors [39] Cell adhesion and morphology are dynamically mut- able during cell migration [22]. The last issue that we addressed in our work is thus the free motility of micro- glia in culture. While numerous publications cite motil- ity and short migration of microglia in vivo, only few works describe microglia moving for long distances. This is the case for example of the work by Carbonell and co- authors [34], demonstrating by intracerebroventricular injection of rhodamine and time-lapse confocal micros- copy that subventricular microglia at the interface of the cerebrospinal fluid and brain parenchyma, exhibit the “in situ” ability to migrate for several hundred microns into the parenchyma, towards a deafferentation injury of the hippocampus. Conversely, Nimmerjahn and colleagues in- dicate only static movement of microglia, branch motility, but not cell bodies migration [2]. Discussion Moreover, future analysis of parameters such as cell Moreover, by the use of bio-mimetic 3D microfluidic substrates, our work demonstrates that primary micro- glia appear intrinsically different from immortalized N9 cells. During cell polarization inside the microchannels, primary microglia tend to align longitudinally and indi- vidually, whereas immortalized N9 microglia assume a more compact morphology with the propensity to es- tablish cell contacts. This might be reminiscent of the immortalization program that likely confers to N9 cells a compact morphology optimizing cell division and Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 8 of 12 Figure 8 Morphometric analysis of primary microglia migrating into microfluidic channels. A. Mean accumulated distance of microglia somata over 20 h time-lapse recording. B. Mean velocity (μm/min) of microglia somata in microfluidic devices. C. Randomly selected cells moving in the microchannels (n = 79 cells) and culturing chamber (n = 120 cells) are manually tracked. Image stacks and time series are analyzed by Image J software. Data presented as mean ± SD. Statistical difference is obtained by Mann–Whitney test (p < 0.0003). “M” is major axis, “m” minor axis, “P” cell perimeter, “A” cell area, “AR” aspect ratio, “CR” circularity ratio. Figure 8 Morphometric analysis of primary microglia migrating into microfluidic channels. A. Mean accumulated distance of microglia somata over 20 h time-lapse recording. B. Mean velocity (μm/min) of microglia somata in microfluidic devices. C. Randomly selected cells moving in the microchannels (n = 79 cells) and culturing chamber (n = 120 cells) are manually tracked. Image stacks and time series are analyzed by Image J software. Data presented as mean ± SD. Statistical difference is obtained by Mann–Whitney test (p < 0.0003). “M” is major axis, “m” minor axis, “P” cell perimeter, “A” cell area, “AR” aspect ratio, “CR” circularity ratio. Figure 8 Morphometric analysis of primary microglia migrating into microfluidic channels. A. Mean accumulated distance of microglia somata over 20 h time-lapse recording. B. Mean velocity (μm/min) of microglia somata in microfluidic devices. C. Randomly selected cells moving in the microchannels (n = 79 cells) and culturing chamber (n = 120 cells) are manually tracked. Image stacks and time series are analyzed by Image J software. Data presented as mean ± SD. Statistical difference is obtained by Mann–Whitney test (p < 0.0003). “M” is major axis, “m” minor axis, “P” cell perimeter, “A” cell area, “AR” aspect ratio, “CR” circularity ratio. Discussion Using transcranial two photon microscopy, Davalos and coauthors [8] confirm that only microglial processes are highly dynamic in intact Page 9 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 with line-grating and pillar-shaped geometries. The micropatterns dimensions in both cases are width: 1500 nm, pitch: 3 μm, height: 550 nm. The fabrication process is started with the patterning of PMMA by 100 kV e-beam lithography on a Si substrate. A 20 nm Cr film is evapo- rated by electron gun followed by lift-off process in acetone at 50°C and sonication. Samples are then etched up to 550 nm by Reactive Ion Etching using CHF3, O2, SF6 and Ar gas mixtures. After Cr wet etching, Si substrates are thermally oxidized at 950°C for 2 h in O2 atmos- phere to reduce surface roughness and then immersed in the hydrofluoric acid buffer to remove the thin oxide layer. After cleaning in Piranha solution (H2SO4/H2O2, 3:1), microstructured Si wafers are silanized with 10% trimethylchlorosilane in toluene in N2 environment to generate a low-energy surface and facilitate the subse- quent mold-PDMS separation. The micropatterns are reproduced on PDMS by standard soft lithographic techniques. A 10:1 (v/v) mixture of monomer and cur- ing agent is prepared and diluted in a 5% (v/v) solution in n-heptane, in order to lower the viscosity. The mix- ture spin-coated onto the Si master mold is left undis- turbed for 2–3 h to allow the solution to penetrate into the voids of the master and for solvent evaporation. Then, it is cured for 30 min at 60°C. Onto this thin PDMS layer, a liquid prepolymer of Sylgard 184 PDMS 10:1 (v/v) is poured and degassed. Then, it is reticulated on a hotplate for 1 h at 60°C and peeled off from the mold. Micropatterned PDMS samples are plasma oxi- dized and kept in water before cell culture, in order to produce and maintain a hydrophilic surface. prove that the leading edge of mouse primary microglia moves for a maximal displacement of 50 and 120 μm after 30 minutes, in a gradient of ATP or ADP, respectively. However, the average distance spontaneously migrated in the absence of any provided stimulus is only 0.8 μm. All considered, spontaneous long distance migration of microglia is described in vivo with conflicting results, but never in vitro until now. Conclusions With our work we have shown that a strict control over biomaterial surface topography by soft lithography tech- niques can highly impact on microglia morphology and greatly improve spontaneous motility in vitro. The use of microstructured 3D devices to manipulate microglia is thus of interest to scientists working on the mechanisms of cell substrate/matrix interactions, morphogenesis and migra- tion, and particularly on microglia responses and functions also during neurodevelopmental and neuroinflammatory disorders. The advantage of using primary microglia in microfluidics and micropatterning opens the road to the use of these devices for testing drug candidates for CNS neuroinflammatory diseases. Reagents All reagents for cell culture are obtained from Sigma- Aldrich, unless otherwise stated. The culture media DMEM and DMEM-F12 are acquired from Invitrogen. Fetal bovine serum (FBS) is obtained from Gibco. Discussion Here, we demonstrate that biocompatible polimeric channels constitute a more suited environment than transwell devices or Dunn chemotaxis chambers for permitting and measuring spontaneous long distance migration in vitro. Indeed, the 500 μm traveled by primary microglia inside the microfluidic channels in the absence of stimuli are much above the known average distance so far reported and this achievement might be perhaps improved. In summary, by the use of interdisciplinary techniques, our data indicate that microfluidic technologies and microstructured patterns with control over the presenta- tion of adhesion sites, are able to sort out different mor- phological structures within a mixed microglia population and allow free motility in vitro. By reproducing a special- ized niche for the cells, these technologies can help to understand the role of structural determinants in priming morphogenesis and free motility of microglia and may be exploited for translational research on functional tissue engineering and implantable device design. Fabrication and design of microfluidic devices for real- time cell analysis Standard soft lithography procedures are performed to fabricate all microdevices in PDMS, a biocompatible thermo-curable elastomer [40,41]. The microfluidic device features two cell culture compartments (1 mm wide, 7 mm in length and 100 μm high) connected via a set of micron-size channels each with dimensions: width = 12 or 18 μm (depending on the experiment), length = 500 μm, height = 10 μm (Figure 4). The circular wells (8 mm in diameter) serve as loading inlets and cell medium reser- voirs for nutrient and gas exchange. The design configur- ation was adapted from Hosmane and collaborators [42], optimizing microchannel dimensions to the microglia physical scale. The master molds are realized by two-layer microfabrication process using the negative photoresist SU-8 (MicroChem Corp, Newton, MA). Briefly, patterns for standard photolithography are designed with CAD software and transferred on two chrome masks by electron-beam lithography. Silicon wafers are spin coated with a layer of SU-8 3005 at a rate of 1000 rpm (resist thickness 10 μm), pre-baked at 95°C for 3 min, exposed to Fabrication and preparation of microstructured PDMS substrates Microtopographic silicon masters are realized to replica- mold substrates on PDMS (Sylgard 184, Dow Corning) Page 10 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 10 of 12 laws (D.L. 116/92). The ethical procedure has been ap- proved by the Animal Welfare Office, Department of Public Health and Veterinary, Nutrition and Food Safety, General Management of Animal Care and Veterinary Drugs of the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the num- ber of animals only necessary to produce reliable results. Primary microglial cultures are prepared from 1 to 2 day-old rat and mouse, as previously described by Chen and collaborators [44]. In brief, after removing the meninges, cortices are minced and digested with 0.01% trypsin and 10 μg/ml DNase I. After dissociation and passage through 70-μm nylon cell strainer (BD Biosci- ences Europe), cells are resuspended in DMEM medium supplemented with 20% heat-inactivated FBS, 4 mM glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml gentamicin and plated in T75 poly-D-lysine-coated flasks, at about 10 million cells/flask. The cultures are kept at 37°C in a 5% CO2 and 95% air atmosphere. Every 2–3 days, the medium is en- tirely changed for the next 12-days. At about 14 days after plating, mixed glial cultures are shaken at 200 rpm at 37°C for 1 hour. The microglial cells are collected from each flask and plated at different density on microfluidic de- vices or micropatterned supports coated with 10 μg/ml fi- bronectin. A population 99% pure of microglial cells is obtained as verified by immunofluorescence with GFAP (for astrocytes), NeuN (for neurons), NG2 (for oligoden- drocytes) and CD11b clone OX42 (for microglia). a i-line (365 nm) UV light source for 16 s through a photo mask (with the microchannel pattern and alignment marks) and post-baked (1 min at 65°C, 3 min at 95°C). Next, the second photolithography step on Su-8 3050 (100 μm thick) transfers the chamber areas and reservoirs aligned to the first pattern (pre-bake: 45 min at 95°C, ex- posure time: 18 seconds, post-bake: 1 min at 65°C, 5 min at 95°C). PDMS (Sylgard 184, Dow Corning) chips are obtained by replica molding, casting the prepolimer base and cross-linker at the volume ratio of 10:1, over the pat- terned master template. Coating of microfluidic devices with fibronectin g After UV sterilization for 20 min, the coating of micro- fluidic devices is performed according to Park and coau- thors [43]. Each reservoir is loaded with 100 μl of 10 μg/ ml fibronectin (Sigma-Aldrich) and the entire device is allowed to be filled. The coating solution is kept for 1 h at room temperature and three washes are performed after removal of the fibronectin solution. Homogeneity of fibronectin coating is verified by indirect immunofluor- escence, in the presence of anti-fibronectin (Calbiochem) used at a dilution of 1:100 in phosphate buffer saline (PBS, 24 h at 4°C), followed by rabbit anti-goat rhodamine conjugated antiserum (3 h at 24°C). Confocal analysis is performed (as described below), and the profile of fluores- cence intensity is obtained with the Zen software of Zeiss LSM 700 microscope. Cell loading Loading of the cells in the microfluidic devices is performed mainly according to Park and coauthors [43]. Briefly, the cell device is maintained filled with 200 μl of culture medium for 1 h in a humidified incubator, before plating the cells. The medium is then removed from the reservoirs and 1×104 cells are immediately seeded. The device is kept in the incubator for 15–20 min to allow cell adhesion and, after replacement of fresh media in the reservoirs, the device is then maintained at 37°C in a 5% CO2 and 95% air atmosphere, until used. Plating density is set in the range of 125 cells/mm2. Fabrication and preparation of microstructured PDMS substrates After degassing for 30 minutes in a vacuum chamber the PDMS is allowed to polymerize at 120°C on a hotplate for 1 h. Once cross-linked, it is care- fully released from the mold and then fluidic access ports are created using a suite of 8 mm dermal biopsy punch tools (Kai Medical). To form an irreversible bonding, the surfaces of PDMS replica and microscope glass slides are O2 plasma-activated (Oxford Plasma Lab 80 plus, RF Power: 20 W, Flux: 60 sccm, Pressure: 700 mtorr, Time: 30 s) and put in contact immediately after exposure. Assembled devices are post-baked at 70°C for 2 h to complete and enhance adhesion strength. Before plasma treatment bonding, microscope glass slide (52 mm × 76 mm, Menzel-Glaser) are cleaned in Piranha solution (H2SO4/H2O2 3:1), rinsed in DI water and dried with N2 gun to remove debris and other surface contaminants. Coating of micropatterned structures with fibronectin After UV sterilization for 20 min, the micropatterned devices are kept immersed in a solution of 10 μg/ml fi- bronectin (Sigma-Aldrich) for 1 h at room temperature, followed by three washes with sterile water. Microglial N9 cell line Microglial N9 cell line The murine N9 microglia cell line is grown in DMEM-F12 medium supplemented with 10% heat-inactivated FBS, 4 mM glutamine, 50 U/ml penicillin, 50 μg/ml strepto- mycin and 100 μg/ml gentamicin. The N9 microglia is kept at 37°C in a 5% CO2 and 95% air atmosphere. Statistical analysis The Mann–Whitney test is used for non-parametric ana- lysis of differences between groups. P <0.05 is considered statistically significant. Primary cortical microglia Microglia are washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.05-0.1% Triton X-100 for 10 min, rinsed, blocked All animal procedures have been performed according to the European Guidelines for the use of animals in re- search (86/609/CEE) and the requirements of Italian Page 11 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 11 of 12 for 30 min in 1% PBS/BSA (bovine serum albumin), and stained with 5 μg/ml Cy2-phalloidin (Sigma-Aldrich) alone, or in combination with the primary antiserum against P2Y12 receptor (Anaspec) used at 1:100 diluition, in 1% PBS/BSA, for about 3 h at 37°C, or stained with anti- paxillin (BD Biosciences) used at 1:1000. The secondary antibodies used for double labeling are Cy3-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunoresearch) or Cy2-conjugated donkey anti-mouse IgG (1:100, Jackson Immunoresearch). The cells are then extensively washed and stained with the nucleic acid blue dye, Hoechst 33342 (1:1000). After rinsing, the cells are covered with gel/ mount™anti-fading medium (Biomeda Corporation) and a coverslip. Incorporated fluorescence is analyzed by means of a fluorescence microscope (Olympus BX51). Brightness and contrast of digital images are adjusted using Microsoft Office PowerPoint 2007. morphometric parameters. Tracking analysis of time-lapse microphotographs is performed by Image J manual track- ing plugin. The generated tracking data are used to calcu- late microglia motility parameters. Audio Video Interleave (AVI) files are generated by using the entire time-lapse image sequence with Image J software (Additional file 1). Immunofluorescence using microfluidic devices u o uo esce ce us g c o u d c de ces Microglia is washed three times by loading each reser- voir with 200 μl PBS. Cells are then fixed with 4% para- formaldehyde for 20 min, washed, permeabilized with 0.05-0.1% Triton X-100 for 15 min, rinsed, blocked for 30 min in 1% PBS/BSA, and stained with 5 μg/ml Cy2- phalloidin (Sigma-Aldrich), alone or in combination with the primary antiserum against P2Y12 receptor (Anaspec) used at 1:100 dilution, or with IBA1 (Wako Chemicals GmbH) at 1:200, in 1% PBS/BSA, for 24 h at 4°C. The secondary antibody used for double immunofluores- cence is Cy3-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunoresearch). Finally, the cells labeled with IBA1 are also extensively washed and allowed to incorp- orate the nucleic acid blue dye, Hoechst 33342 (1:1000). Confocal microscopy After rinsing of the cells, double or triple incorporated im- munofluorescence is analyzed by means of a confocal laser scanning microscope (LSM 700, Zeiss) equipped with four laser lines: 405 nm, 488 nm, 561 nm and 639 nm. The brightness and contrast of the digital images are adjusted using Microsoft Office PowerPoint 2007. Competing interests The authors declare that they have no competing interests. he authors declare that they have no competing interests. Received: 25 March 2013 Accepted: 3 October 2013 Published: 13 October 2013 Received: 25 March 2013 Accepted: 3 October 2013 Published: 13 October 2013 Acknowledgments We thank Dr. N. D’Ambrosi and Dr. S. Apolloni for useful advice and help with primary cultures preparation. This study was supported by grant from Ministero della Salute (GR-2009-1523273). References 1. del Rio-Hortega P: Microglia. In Cytology and Cellular Pathology of the Nervous System. Edited by Penfield W. New York: Hoeber; 1932:482–534. Abbreviations A C ll AR A: Cell area; AR: Axis ratio; BSA: Bovine serum albumin; CNS: Central nervous system; CR: Circularity ratio; 3D: Three-dimensional; FBS: Fetal bovine serum; M: Major axis; m: minor axis; P: Cell perimeter length; PBS: Phosphate buffer saline; PDMS: Poly(dimethylsiloxane). Authors’ contributions CV and LB conceived and designed the study. LB and AG designed microstructured PDMS substrates and microfluidic devices for cell culturing. AD provided fabrication and preparation of microstructured PDMS substrates and microfluidic devices. SA performed primary microglia purification, culturing on microstructured substrates and microfluidic devices, and real time cell analysis. SA and CM completed confocal fluorescence analysis. LB, AD and AG gathered and analyzed data. CV and SA wrote the article, which was revised by LB and AG. All authors read and approved the final version of manuscript. Author details 1 1Santa Lucia Foundation/CNR-Cellular Biology and Neurobiology Institute, Via del Fosso di Fiorano 65, 00143 Rome, Italy. 2Department of Anatomy, Histology, Forensic Medicine and Orthopedics, University La Sapienza, Rome, Italy. 3CNR-Institute for Photonics and Nanotechnology, Via Cineto Romano 42, 00156 Rome, Italy. Additional file Additional file 1: The video shows baseline motility of primary microglia for long distances on artificial bio-mimetic microfluidic substrates with controlled physical/chemical characteristics. We Additional file 1: The video shows baseline motility of primary microglia for long distances on artificial bio-mimetic microfluidic substrates with controlled physical/chemical characteristics. We performed regular 30 min time-lapse recording (JuLI, Digital Bio) over a 20 h period. Primary microglia spontaneously travel through fibronectin- coated PDMS microfluidic channels (width 12 μm and length 500 μm) for a distance of up to 500 μm in approximately 12 h, with an average speed of 0.66 μm/min. Additional file 1: The video shows baseline motility of primary microglia for long distances on artificial bio-mimetic microfluidic substrates with controlled physical/chemical characteristics We substrates with controlled physical/chemical characteristics. We performed regular 30 min time-lapse recording (JuLI, Digital Bio) over a 20 h period. Primary microglia spontaneously travel through fibronectin- coated PDMS microfluidic channels (width 12 μm and length 500 μm) for a distance of up to 500 μm in approximately 12 h, with an average speed of 0.66 μm/min. Time-lapse microscopy Primary microglia are recorded with objective 4× at regular time-lapse (30 min) over a 20 h period, with a bright-field microscope equipped by a time-lapse system (JuLI, Digital Bio). 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https://openalex.org/W4200622023	https://www.frontiersin.org/articles/10.3389/froh.2021.769988/pdf	English	null	Data_Sheet_3.PDF	null	2,021	cc-by	7,511	Connecting Medical Personnel to Dentists via Teledentistry in a Children’s Hospital System: A Pilot Study Kimberly J. Hammersmith 1,2, Macaire C. Thiel 1,2, Matthew J. Messina 2, Paul S. Casamassimo 1,2 and Janice A. Townsend 1,2 Kimberly J. Hammersmith 1,2, Macaire C. Thiel 1,2, Matthew J. Messina 2, Paul S. Casamassimo 1,2 and Janice A. Townsend 1,2 1 Nationwide Children’s Hospital, Columbus, OH, United States, 2 The Ohio State University College of Dentistry, Columbus, OH, United States Investigators evaluated feasibility, acceptability, and sustainability of a teledentistry pilot program within a children’s hospital network between March, 2018, and April, 2019. The program connected dentists to medical personnel and patients being treated in urgent care clinics, a primary care clinic, and a freestanding emergency department via synchronous video consultation. Three separate but parallel questionnaires evaluated caregiver, medical personnel, and dentist perspectives on the experience. Utilization of teledentistry was very low (2%, 14/826 opportunities), but attitudes regarding this service were largely positive among all groups involved and across all survey domains. Uptake of new technology has barriers but teledentistry may be an acceptable service, especially in the case of dental trauma. Investigators evaluated feasibility, acceptability, and sustainability of a teledentistry pilot program within a children’s hospital network between March, 2018, and April, 2019. The program connected dentists to medical personnel and patients being treated in urgent care clinics, a primary care clinic, and a freestanding emergency department via synchronous video consultation. Three separate but parallel questionnaires evaluated caregiver, medical personnel, and dentist perspectives on the experience. Utilization of teledentistry was very low (2%, 14/826 opportunities), but attitudes regarding this service were largely positive among all groups involved and across all survey domains. Uptake of new technology has barriers but teledentistry may be an acceptable service, especially in the case of dental trauma. Edited by: Edited by: Ramprasad Vasthare Prabhakar, Manipal College of Dental Sciences, Manipal, India Reviewed by: Balgis Gaffar, Imam Abdulrahman Bin Faisal University, Saudi Arabia Julia Chang, National Taiwan University, Taiwan Keywords: teledentistry, emergency department, urgent care, dentistry, dental trauma National Taiwan University, Taiwan Correspondence: Kimberly J. Hammersmith kim.hammersmith@ nationwidechildrens.org INTRODUCTION Over the last two decades, increasing numbers of pediatric patients have sought care at emergency departments (ED), urgent care (UC) facilities, and medical clinics with oral complaints including toothaches, dental trauma, and other concerns [1]. Medical providers have little oral health training and education [2]. Treatment for dental problems by medical providers is limited and may be managed by ineﬀective prescriptions for opioids or antibiotics. Patients receiving oral health care in an ED are more than 7 times more likely to receive an opioid prescription than patients treated in a dental oﬃce [3]. About 65% of patients receive antibiotics for their dental problem from medical providers when the indicated treatment for their condition is a procedure performed by a dentist [4]. Challenges related to low socioeconomic status, inadequate access to dentists particularly in rural areas, and low oral health literacy contribute to patients seeking dental care in ED or other medical facilities [5]. Specialty section: This article was submitted to Oral Health Promotion, a section of the journal Frontiers in Oral Health Received: 03 September 2021 Accepted: 15 November 2021 Published: 09 December 2021 BRIEF RESEARCH REPORT published: 09 December 2021 doi: 10.3389/froh.2021.769988 Citation: Hammersmith KJ, Thiel MC, Messina MJ, Casamassimo PS and Townsend JA (2021) Connecting Medical Personnel to Dentists via Teledentistry in a Children’s Hospital System: A Pilot Study. Front. Oral. Health 2:769988. doi: 10.3389/froh.2021.769988 Inappropriate care seeking at medical facilities for oral concerns can be addressed by diﬀerent interventions. More than half of patients arrive during business hours when dental oﬃces are normally open [6]. Programs diverting patients from ED and UC centers and connecting them with dentists are one option [7]. Telehealth is another possible adjunct to provide guidance for patients physically located in medical settings while simultaneously exposing medical personnel to best practices from a dentist. December 2021 \| Volume 2 \| Article 769988 Frontiers in Oral Health \| www.frontiersin.org Physicians to Dentists via Teledentistry Hammersmith et al. volumes of oral-related diagnostic codes were added to the same protocol to increase encounters for a total of ﬁve sites. We focused our data analysis once all ﬁve sites were included, for a period of 5 months. Telemedicine, connecting medical providers with other medical providers, has been feasible and acceptable for eﬀorts in underserved areas [8] and improves the quality of care for patients with chronic conditions [9–12]. Additionally, telemedicine consultations with specialists produces better outcomes during acute or emergency care [13–16]. A 2017 study showed that mental health and primary care were the most common forms of telemedicine [17]. This study to evaluate the pilot program was approved as exempt from the Nationwide Children’s Hospital Institutional Review Board. Data from electronic medical records for all ﬁve sites was gathered including patient demographics, encounter information, and ICD-10 codes (Table 1) involving oral- or dental-related chief complaints. Barriers to successful implementation of telemedicine programs include state medical and dental licensing restrictions, inadequate reimbursement, and prohibitive legislative policy [18, 19]. Suboptimal ﬁnancial reimbursement may hinder widespread adoption and sustainability of telemedicine [8]. All ﬁfty states provide some Medicaid reimbursement for telehealth services, but diﬀer dramatically in deﬁning and regulating telehealth, so reimbursement may not meet expense [20]. Live, synchronous video visits are reimbursed the highest; asynchronous modalities often have restrictions. Some states put restrictions on eligible providers, eligible facilities, or require speciﬁc informed consent requirements [20]. Three separate surveys were developed with duplicate or similar questions when possible to evaluate the teledentistry program as to its acceptability, feasibility, and sustainability. RESULTS During the pilot period of 5 months across the ﬁve sites, 826 patients presented with an oral or dental complaint with average of 165 patients a month, though some would have been excluded from participating in teledentistry due to language barriers. A dentist was only consulted via teledentistry 14 times or <2% of possible encounters. During the pilot, the chief complaint of dental trauma comprised 5% of total visits but 50% of the teledentistry encounters (Table 1). A chief complaint of a lesion, cyst, or pathology occurred in 266 (31%) of dental-related encounters but resulted in zero teledentistry consults. Frontiers in Oral Health \| www.frontiersin.org Citation: Surveys for the caregivers, the medical personnel initiating the teledentistry consultation, and the consulting dentist were adapted from models in the literature [25, 26]. Survey domains included: connection and equipment, comfort, competence of provider, teledentistry as substitute for in-person examination, access, workﬂow and eﬃciency, and preference. Following demographic questions gauging age, role, and experience with telemedicine technology, medical personnel answered 15, 5-point Likert scale type questions (14 such questions on the dentist survey). The survey intended for caregivers consisted of 15, 5-point Likert scale type questions and 4 questions had three answer choices. Teledentistry is the use of teleconferencing software and cameras that allow a dentist to evaluate a patient remotely [21]. Cameras and software allow a live feed conference, synchronous consultation, or saving of images to be evaluated later, called asynchronous consultation [21]. Teledentistry was utilized by the Department of Defense in the 1990s in a pilot project aimed at patient care, continuing education, and dentist- laboratory communication [22]. Currently sixteen state laws include language speciﬁc to teledentistry [20]. The caregiver accompanying each child received a survey directly following a teledentistry encounter, while still in the ED. Exclusion criteria were caregivers who did not consent or were non-English speaking. Surveys were administered via paper for caregivers and REDCap [27], a secured web application, for dentists and medical personnel, who completed the surveys electronically soon after the encounter. Data was assessed using descriptive statistics only. Caregivers received a gift card as an incentive for completing the survey. Many examples of telemedicine connecting medical professionals to other medical professionals exist, but no studies have examined use of teleconsultation between dental and medical professionals. Only one study has suggested it as a means to improve interdisciplinary communication [23]. In late 2018, Ohio passed legislation that allows remote, synchronous consultations by a dentist to be recognized and billed similarly to an in-person encounter [24]. The aim of this study was to evaluate the feasibility, acceptability, and sustainability of a pilot teledentistry program in a children’s hospital network in Ohio. MATERIALS AND METHODS FIGURE 1 \| Location of patients (%) with oral or dental chief complaint. 14 surveys, from 11 unique respondents), and dentist (n = 14 surveys from 7 unique respondents) Medical personnel camera was considered easy by most dentists, but in 3 encounters (21%) the dentist found it diﬃcult Comments indicated the TABLE 1 \| Frequency of dental diagnosis codes at ﬁve medical sites during pilot period. Pilot period encounters, N = 826 Teledentistry encounters, N = 14 ICD-10 diagnosis codes Description N Percent % N Percent % K00.1, K09.0, K09.8, K12.0, K12.1, K12.30, K13.0, K13.21, K13.4, K13.70, K13.79 Lesions, cysts, pathology 266 32 0 0 K04.7, K12.2, L03.211, L03.213 Cellulitis or infections 151 18 3 21 K00.2, K02.9, K08.89, K08.9 Caries and tooth disorders 153 19 3 21 K00.6, K00.7 Teething and eruption 134 16 0 0 K05.00, K05.5, K05.6, K05.10, K05.219, K06.1, K06.8, K06.9 Gingivitis and periodontal disease 73 9 1 7 K03.81, K08.109, S02.5XXA, S02.5XXB, S03.2XXA, S03.2XXD Trauma 43 5 7 50 K11.1, K11.20, K11.21, K11.6, K11.7, K11.8 Salivary glands 31 4 0 0 Total 851 100 14 100 851 codes noted, with 826 individual patients, as some patients received multiple diagnostic codes during their encounters. TABLE 1 \| Frequency of dental diagnosis codes at ﬁve medical sites during pilot period. FIGURE 1 \| Location of patients (%) with oral or dental chief complaint. FIGURE 1 \| Location of patients (%) with oral or dental chief complaint. 14 surveys, from 11 unique respondents), and dentist (n = 14 surveys, from 7 unique respondents). Medical personnel surveyed included 5 registered nurses and 6 physicians with some repeated, or non-unique, users. The majority were 31–50 years old, 7 (55%) were using teledentistry for the ﬁrst time, and 11 (92%) had no previous experience using telemedicine technology for other services. camera was considered easy by most dentists, but in 3 encounters (21%), the dentist found it diﬃcult. Comments indicated the need for better training of medical personnel on use of the intraoral camera (Table 3). All dentists but one (93%, n = 13) felt the software was easy to use and just over half (58%, n = 8) of dentists felt they could hear and visually assess the patient’s area of concern as well as if they were present. Frontiers in Oral Health \| www.frontiersin.org MATERIALS AND METHODS Nationwide Children’s Hospital in Columbus, OH, USA, launched a pilot teledentistry program in March 2018, connecting a free-standing, suburban ED with its dental department. The ED was equipped with a cart with a laptop, external speaker, webcam, and intraoral camera. Two dentists (MT and KH) trained the staﬀon teledentistry protocol and how to use equipment. When a patient presented with an oral- or dental-related chief complaint, the ED provider was to order a consultation with the on-call dental resident who would be paged, and then connect to the ED via a secure, synchronous platform HealthChat R⃝(Miami, FL, USA). The dentist could then communicate directly with patient and family as well as ED staﬀregarding care. During the pilot, the majority of teledentistry consults originated in one of the UC sites (50%), although patients with dental diagnosis codes arrived at all ﬁve sites (Figure 1). There were zero encounters from the primary care site. The disposition of all patients with a dental diagnosis code was 98% discharged, and 2% transferred to the main ED. Of the 14 teledentistry patients, 79% were discharged, and 21% were transferred. Due to low utilization of teledentistry by the ED in the ﬁrst 8.5 months, three UC sites and one primary care site with higher Ten encounters included survey data from all three involved parties: patient/family (n = 14), medical personnel (n = December 2021 \| Volume 2 \| Article 769988 2 Physicians to Dentists via Teledentistry Hammersmith et al. Hammersmith et al. Physicians to Dentists via Teledentistry TABLE 1 \| Frequency of dental diagnosis codes at ﬁve medical sites during pilot period. Pilot period encounters, N = 826 Teledentistry encounters, N = 14 ICD-10 diagnosis codes Description N Percent % N Percent % K00.1, K09.0, K09.8, K12.0, K12.1, K12.30, K13.0, K13.21, K13.4, K13.70, K13.79 Lesions, cysts, pathology 266 32 0 0 K04.7, K12.2, L03.211, L03.213 Cellulitis or infections 151 18 3 21 K00.2, K02.9, K08.89, K08.9 Caries and tooth disorders 153 19 3 21 K00.6, K00.7 Teething and eruption 134 16 0 0 K05.00, K05.5, K05.6, K05.10, K05.219, K06.1, K06.8, K06.9 Gingivitis and periodontal disease 73 9 1 7 K03.81, K08.109, S02.5XXA, S02.5XXB, S03.2XXA, S03.2XXD Trauma 43 5 7 50 K11.1, K11.20, K11.21, K11.6, K11.7, K11.8 Salivary glands 31 4 0 0 Total 851 100 14 100 *851 codes noted, with 826 individual patients, as some patients received multiple diagnostic codes during their encounters. MATERIALS AND METHODS The majority of caregivers (92%, n = 11) felt the dentist could hear and see their child’s problem as well as if in the room. Table 2 displays the questions asked across all domains in each survey, as well as the overlap of questions across survey respondents. All medical personnel felt videoconferencing software and maneuvering the intraoral camera were easy. Directing and instructing medical personnel to use the intraoral Regarding the comfort domain, most medical personnel (92%, n = 11) and dentists (93%, n = 13), as well as 100% (n = 12) of caregivers were comfortable with the process. All caregivers December 2021 \| Volume 2 \| Article 769988 3 Physicians to Dentists via Teledentistry Hammersmith et al. TABLE 2 \| Selection of teledentistry survey questions by domain. Workﬂow and efﬁciency I am satisﬁed with how long I got to talk to the dentist on the computer I am satisﬁed with how long I got to talk to the dentist on the computer SA: 67%, A: 33%, N: 0%, D: 0%, SD: 0% I am satisﬁed with how long I waited to talk to the dentist SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% Talking with the dentist on the computer today was worth the time that I saved in not having to go to Nationwide Children’s main emergency department downtown SA: 50%, A: 17%, N: 33%, D: 0%, SD: 0% N/A I am satisﬁed with how long I got to talk to the dentist on the computer SA: 67%, A: 33%, N: 0%, D: 0%, SD: 0% I am satisﬁed with how long I waited to talk to the dentist SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% Talking with the dentist on the computer today was worth the time that I saved in not having to go to Nationwide Children’s main emergency department downtown SA: 50%, A: 17%, N: 33%, D: 0%, SD: 0% N/A I would prefer to consult patients similar to this patient via teledentistry rather than in person SA: 43%, A: 43%, N: 7%, D: 7%, SD: 0% N/A surveyed said that teledentistry was explained and they knew what to expect, and all but one (91%, n = 11) agreed their personal information was protected during the interaction with the dentist. However, most dentists (85%, n = 12) disagreed that patient satisfaction would be the same if still photos were used rather than video. When asked if the intraoral camera view of the patient’s problem were equivalent to an in-person assessment, only 2 (14%) dentists disagreed. Most caregivers (83%, n = 10) and all dentists (100%, n = 14) felt the patient received the same recommendations as if the dentist were present. When caregivers were asked if a medical provider could have managed alone, without contacting the dentist, 3 (25%) agreed, 6 (50%) were neutral, and 3 (25%) disagreed. As far as provider competence, all medical personnel (n = 12) thought the dentist understood the patient’s problem. Thirteen dentists (93%) felt able to gather necessary information to make a conﬁdent diagnosis or recommendation, as they would with a non-teledentistry patient. Frontiers in Oral Health \| www.frontiersin.org MATERIALS AND METHODS Patient Medical personnel Dentist Connection and equipment N/A Using the videoconferencing software (Healthchat) was: Using the videoconferencing software (Healthchat) was: VE: 33%, E: 42%, N: 25%, D: 0%, VD: 0% VE: 43%, E: 50%, N: 0%, D: 7%, VD: 0% N/A Maneuvering the intraoral camera for live video was: Directing and instructing medical staff to use the intraoral camera was: VE: 42%, E: 50%, N: 8%, D: 0%, VD: 0% VE: 7%, E: 57%, N: 14%, D: 21%, VD: 0% N/A The instructions for connecting to Healthchat were clear and accurate The instructions for connecting and conducting a consult via Healthchat were clear and accurate SA: 50%, A: 42%, N: 8%, D: 0%, SD: 0% SA: 43%, A: 57%, N: 0%, D: 0%, SD: 0% I think the dentist could hear and see my child’s problem as well as if the dentist were in the room N/A I could hear and visually assess the patient’s area of concern as well as if I were there in person SA: 58%, A: 33%, N: 8%, D: 0%, SD: 0% SA: 29%, A: 29%, N: 29%, D: 14%, SD: 0% Comfort I was comfortable with the teledentistry process I was comfortable with the teledentistry process I was comfortable with the teledentistry process SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% SA: 42%, A: 50%, N: 8%, D: 0%, SD: 0% SA: 50%, A: 43%, N: 0%, D: 7%, SD: 0% Someone explained to me that we were going to use teledentistry so that I knew what to expect N/A N/A Yes: 100%, No: 0% I feel that my personal information was protected during the interaction with the dentist N/A N/A SA: 83%, A: 8%, N: 8%, D: 0%, SD: 0% Competence of provider The dentist understood my child’s problem I think the dentist understood the patient’s problem I was able to gather necessary information to make a recommendation or diagnosis I felt comfortable with, as I would with a non-teledentistry patient SA: 75%, A: 17%, N: 8%, D: 0%, SD: 0% SA: 83%, A: 17%, N: 0%, D: 0%, SD: 0% SA: 50%, A: 43%, N: 0%, D: 7%, SD: 0% The dentist responded to my concerns N/A N/A SA: 67%, A: 25%, N: 8%, D: 0%, SD: 0% The dentist explained what’s going on with my child’s teeth or mouth N/A N/A SA: 58%, A: 25%, N: 17%, D: 0%, SD: 0% Teledentistry as substitute for in-person examination My child was given the same recommendations as if the dentist were here in person N/A The patient was given the same recommendations as if I were there in person SA: 66%, A: 17%, N: 17%, D: 0%, SD: 0% SA: 71%, A: 29%, N: 0%, D: 0%, SD: 0% N/A I think still photos rather than video would have allowed the dentist to provide the same diagnosis I think still photos rather than video would have allowed me to provide the same diagnosis SA: 8%, A: 25%, N: 50%, D: 8%, SD: 8% SA: 7%, A: 50%, N: 21%, D: 21%, SD: 0% N/A N/A I think the patient would have the same satisfaction if still photos rather than video were used SA: 0%, A: 0%, N: 14%, D: 71%, SD: 14% The doctor here at this patient care site could have managed my child’s needs ﬁne without getting the dentist on the computer N/A The intraoral camera gave me a view of the problem equivalent to if I were there in person SA: 8%, A: 17%, N: 50%, D: 17%, SD: 8% SA: 14%, A: 64%, N: 7%, D: 14%, SD: 0% Access I would like this patient care site to keep offering this service to patients I would like this patient care site to keep offering this service to other patients I would like this medical location to keep offering this teledentistry service to other patients SA: 92%, A: 8%, N: 0%, D: 0%, SD: 0% SA: 58%, A: 42%, N: 0%, D: 0%, SD: 0% SA: 79%, A: 21%, N: 0%, D: 0%, SD: 0% (Continued) Dentist Using the videoconferencing software (Healthchat) was: SA: 29%, A: 29%, N: 29%, D: 14%, SD: 0% N/A (Continued) Frontiers in Oral Health \| www.frontiersin.org December 2021 \| Volume 2 \| Article 769988 Physicians to Dentists via Teledentistry Hammersmith et al. MATERIALS AND METHODS TABLE 2 \| Continued Medical personnel Patient Patient Had I not had access to the dentist on the computer today, I would not know what to do for my child’s problem SA: 67%, A: 25%, N: 8%, D: 0%, SD: 0% I would recommend the teledentistry process to a friend in a situation similar to mine SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% My insurance should cover this service My insurance should cover this service My insurance should cover this service Yes: 75%, Maybe: 25%, No: 0% MATERIALS AND METHODS TABLE 2 \| Continued Patient Medical personnel Dentist Had I not had access to the dentist on the computer today, I would not know what to do for my child’s problem Had the dentist not been available, the care of this patient would have been compromised N/A SA: 67%, A: 25%, N: 8%, D: 0%, SD: 0% SA: 8%, A: 17%, N: 42%, D: 33%, SD: 0% I would recommend the teledentistry process to a friend in a situation similar to mine I would recommend teledentistry to my colleagues N/A SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% SA: 58%, A: 42%, N: 0%, D: 0%, SD: 0% My insurance should cover this service I think that teledentistry improves the quality of services provided at this patient care site N/A Yes: 75%, Maybe: 25%, No: 0% SA: 67%, A: 33%, N: 0%, D: 0%, SD: 0% Workﬂow and efﬁciency I am satisﬁed with how long I got to talk to the dentist on the computer It takes more than one staff member to use the teledentistry equipment I think that I would be able to treat more patients using teledentistry SA: 67%, A: 33%, N: 0%, D: 0%, SD: 0% SA: 17%, A: 25%, N: 8%, D: 50%, SD: 0% SA: 79%, A: 21%, N: 0%, D: 0%, SD: 0% I am satisﬁed with how long I waited to talk to the dentist The teledentistry consult took an appropriate amount of time N/A SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% SA: 50%, A: 25%, N: 17%, D: 8%, SD: 0% Talking with the dentist on the computer today was worth the time that I saved in not having to go to Nationwide Children’s main emergency department downtown Talking with the dentist on the computer was a good use of the patient’s time N/A SA: 50%, A: 17%, N: 33%, D: 0%, SD: 0% SA: 75%, A: 25%, N: 0%, D: 0%, SD: 0% N/A This consult method ﬁts well into the “process or ﬂow” of this patient care site N/A SA: 50%, A: 33%, N: 17%, D: 0%, SD: 0% Preference In this location, I would prefer: N/A I would prefer to consult patients similar to this patient via teledentistry rather than in person Dentist in person: 33%, No preference: 58%, Dentist on computer: 8% SA: 43%, A: 43%, N: 7%, D: 7%, SD: 0% In this location, I would prefer: N/A N/A Dentist on computer: 33%, No preference: 50%, No dentist available for today’s visit: 17% In this location, I would prefer: N/A N/A Dentist on computer: 33%, No preference: 50%, Transfer to ED downtown: 17% VE, Very easy; E, Easy; N, Neutral; D, Difﬁcult; VD, Very difﬁcult; SA, Strongly agree; A, Agree; N, Neutral; D, Disagree; SD, Strongly disagree. Frontiers in Oral Health \| www.frontiersin.org Workﬂow and efﬁciency They kept asking ’can you see the teeth now?’ But the camera would be showing the ceiling, up the pt’s nose and other parts of the room! Patients seemed to appreciate being able to talk to me directly which is a beneﬁt over still photos. • The amount of time needed was excessive due to technical glitches in the software. Multiple events were created due to staff not being able to see the other person • We had some initial issues with the connection but once that was ﬁgured out it ran very well. The intraoral camera was hard to use at ﬁrst but never used one before. Got easier as time went. This was a great service!! Parents were so appreciative of having the dental consult there and then. Thank you!! personnel indicated they would like the medical site to keep oﬀering teledentistry. two (17%) no dentist at all. Given options of teledentistry or transferring to the main ED, six (50%) caregivers responded neutral, with four (33%) preferring teledentistry and two (17%) transfer to the main ED. As far as dentist preference, 12 dentists (86%) preferred teledentistry to in-person consultation. For workﬂow and eﬃciency, the majority (75%, n = 9) of medical personnel felt the teledentistry encounter took an appropriate amount of time, was a good use of patient’s time (100%, n = 12), and that this consult method ﬁts well into the site process or ﬂow (83%, n=10). Five medical personnel (42%) felt more than one staﬀmember was needed to use the teledentistry equipment. All 14 (100%) dentists thought they would be able to treat more patients using teledentistry. Caregivers were satisﬁed with time available to speak with the dentist (100%, n = 12) and wait time to connect (100%, n = 12). The majority (67%, n = 8) agreed that teledentistry was worth the time saved by not having to go to the main ED that has dental services. Workﬂow and efﬁciency • I think that teledentistry will work very well but the healthcare providers need to be trained in the use of the intraoral camera. I spent around 10 minutes trying to coach the physician to put the software on the correct camera so I could see anything at all. If providers are not tech savvy it may be more useful to have them describe or simply take a picture. • The teledentistry for this patient was very beneﬁcial. It was determined that she needed to be admitted for IV antibiotics. The family didn’t have any transportation to the hospital. They took the COTA bus to our facility. We couldn’t send them by public transportation to the hospital. I feel that with being able to speak with the dentist in real time helped the family understand the need for us to arrange a taxi to take them. The family remained calm even though it took a while to make the arrangements. I feel it was all due to the dentist explaining the complex situation to the family • Camera issues, only able to see via intraoral camera, not chat/videoconferencing camera. Family seemed reassured and glad to have a consult with a dentist. • Having a training session on how to best retract the lips/tongue would be helpful for the physician and perhaps help him/her feel more comfortable when positioning the intraoral camera more comfortable when positioning the intraoral camera • Staff had a difﬁcult time positioning the camera for me to get a diagnostic view. I believe with more practice/training it would be ﬁne. Are medical staff able to see what they are showing the dentist on the intraoral camera? Perhaps they were not looking in the right place but that would be helpful for them. They kept asking ’can you see the teeth now?’ But the camera would be showing the ceiling, up the pt’s nose and other parts of the room! Patients seemed to appreciate being able to talk to me directly which is a beneﬁt over still photos. • Staff had a difﬁcult time positioning the camera for me to get a diagnostic view. I believe with more practice/training it would be ﬁne. Are medical staff able to see what they are showing the dentist on the intraoral camera? Perhaps they were not looking in the right place but that would be helpful for them. Workﬂow and efﬁciency I feel it was all due to the dentist explaining the complex situation to the family • The amount of time needed was excessive due to technical glitches in the software. Multiple events were created due to staff not being able to see the other person • We had some initial issues with the connection but once that was ﬁgured out it ran very well. The intraoral camera was hard to use at ﬁrst but never used one before. Got easier as time went. This was a great service!! Parents were so appreciative of having the dental consult there and then. Thank you!! TABLE 3 \| Comments from caregivers, medical personnel, and dentists. Caregiver Medical personnel Dentists • Some quick training of medical staff regarding how to retract the cheek, lip, or tongue when using the intraoral camera would be helpful • Some quick training of medical staff regarding how to retract the cheek, lip, or tongue when using the intraoral camera would be helpful • Some quick training of medical staff regarding how to retract the cheek, lip, or tongue when using the intraoral camera would be helpful • For some reason the video from the wand with the covering was blurred. This may have been operator (me) error • it was a little time consuming using the Teledentistry software, but I suspect my efﬁciency and ease of use will improve with time • I think this was great - especially for those who live far and don’t have easy access to NCH. It allows for assessment and triaging of true emergencies versus dental problems that can wait until the next day. This will help alleviate the worry parents have as well as freeing up resources in emergency rooms. As far as training - I think it would be beneﬁcial to continue more training with how to maneuver the intraoral camera. This would include retracting the cheek when using the camera. • I think that teledentistry will work very well but the healthcare providers need to be trained in the use of the intraoral camera. I spent around 10 minutes trying to coach the physician to put the software on the correct camera so I could see anything at all. If providers are not tech savvy it may be more useful to have them describe or simply take a picture. Workﬂow and efﬁciency Overall, caregivers felt that the dentist understood their child’s problem (92%, n = 11), and responded to their concerns (92%, n = 11) and the majority agreed the dentist explained their child’s situation (83%, n = 10). As far as access, all 12 medical personnel agreed teledentistry improves the quality of services provided at their site. If the dentist had not been available, 3 (25%) medical personnel thought their patient’s care would have been compromised. All patients and medical personnel would recommend teledentistry in a similar situation. All patients, dentists, and medical Regarding teledentistry as a substitute for in-person examination, some dentists (57%, n = 8) and medical personnel (33%, n = 4) agreed that still photographs would have allowed the dentist to make the same diagnosis as synchronous video. December 2021 \| Volume 2 \| Article 769988 5 Physicians to Dentists via Teledentistry Hammersmith et al. TABLE 3 \| Comments from caregivers, medical personnel, and dentists. Caregiver Medical personnel • It was good and helpful for all the service • 100% Perfect • None, you did great! • It was really kind and helpful for the dentist to help my daughter and check out for her issue or helpful. Really appreciate for the kindness! • For some reason the video from the wand with the covering was blurred. This may have been operator (me) error • it was a little time consuming using the Teledentistry software, but I suspect my efﬁciency and ease of use will improve with time • The ED staff was either intimidated by the technical process or just unwilling to help (I kept hearing ’I’ve never done it’) so I had to take the time to read through the directions while patients were stacking up • The teledentistry for this patient was very beneﬁcial. It was determined that she needed to be admitted for IV antibiotics. The family didn’t have any transportation to the hospital. They took the COTA bus to our facility. We couldn’t send them by public transportation to the hospital. I feel that with being able to speak with the dentist in real time helped the family understand the need for us to arrange a taxi to take them. The family remained calm even though it took a while to make the arrangements. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/froh. 2021.769988/full#supplementary-material ETHICS STATEMENT The studies involving human participants were reviewed and approved by Nationwide Children’s Hospital Institutional Review Board. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. The few encounters limit our ability to report meaningful survey data and associations with variables such as demographics. We had originally intended to compare length of stay, disposition, and patient variables such as age, race, and insurance status for teledentistry and non-teledentistry encounters. Results cannot be tested for statistical signiﬁcance, nor can they be generalized, but they can add to the literature for parties wishing to institute similar programs. ACKNOWLEDGMENTS Mr. Shawn Fallon for his leadership and expertise in teledentistry technology and Dr. Daniel Cohen for his assistance in survey development. CONCLUSIONS provider perception of a larger burden-over-beneﬁt may have contributed to the low number of teledentistry encounters. Importantly, surveys showed that medical personnel’s experiences were generally positive when teledentistry was used, and individual comments about the utility for speciﬁc patients support this sentiment further. Medical personnel largely used teledentistry for patients with dental trauma. Although utilization of teledentistry at medical sites was very low, caregivers, medical personnel, and dentists perceived beneﬁts from using technology in this way. Consulting with a dentist via teledentistry may help medical personnel bridge knowledge and treatment gaps for patients who present to medical clinics with oral or dental concerns. Training medical personnel on using intraoral cameras and teledentistry software can alleviate issues during patient encounters and may increase uptake of new technology. Medical personnel largely used teledentistry for patients with dental trauma. Although utilization of teledentistry at medical sites was very low, caregivers, medical personnel, and dentists perceived beneﬁts from using technology in this way. Consulting with a dentist via teledentistry may help medical personnel bridge knowledge and treatment gaps for patients who present to medical clinics with oral or dental concerns. Training medical personnel on using intraoral cameras and teledentistry software can alleviate issues during patient encounters and may increase uptake of new technology. Additionally, medical providers may perceive they are competent in triaging and managing oral- or dental-related chief complaints in medical settings, resulting in the minimal uptake of teledentistry. Compared to common diagnoses of teething and caries, teledentistry was used disproportionately for trauma codes suggesting medical providers felt less comfortable or familiar with these presentations. As more teledentistry patients were transferred to the main campus ED than non-teledentistry patients (21 vs. 2%), we see that the technology was notably utilized for more severe cases. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Even though medical personnel may have had some issues with the intraoral camera and teledentistry software, the patients liked the technology and appreciated the opportunity to speak to a dental provider when a dentist would otherwise not be accessible. As most answers were positively answered by both dentists and medical personnel, the project appeared to be acceptable. FUNDING Previous research on adoption of teledentistry sheds similar light on the main challenges and best processes for development and implementation steps [28]. While policy and ﬁnancing issues were not obstacles in our pilot, acceptance, perceived usefulness, and ease of use were. As our distribution of consults was not representative of the patient encounters across sites, staﬀat one pilot site may have been more enthusiastic about teledentistry, and interdisciplinary collaborations may beneﬁt from teledentistry champions. With renewed engagement from valuable stakeholders as well as everyday users, additional and continued trainings regarding the intraoral camera and software, teledentistry could be considered more feasible and sustainable in this setting. As the study was conducted prior to the pandemic, similar programs may experience better utilization due to the current urgency and perhaps indeﬁnite rationale behind providing remote care. A Delta Dental Foundation Masters Thesis Award provided funding for printing of the surveys, patient and provider incentives to complete the surveys, and statistical consultation. AUTHOR CONTRIBUTIONS All authors contributed to the design and implementation of the survey and research, to the analysis of the results, and to the writing of the manuscript. DISCUSSION This study is the ﬁrst to describe how teledentistry can be used to facilitate consultation and communication between dentists and medical personnel and revealed barriers in connecting the two. The pilot project utilized live, hands-on trainings at each site, with regular assessments and updates via email. Medical personnel initially expressed interest and enthusiasm for the new technology, but despite coaching, reminders, incentives, and motivation, the pilot utilization of teledentistry technology was extremely low. Regarding preference, when caregivers were given the option between teledentistry and dentist in person, the majority (58%, n = 7) responded neutral, with one (8%) choosing teledentistry. Given the options of teledentistry or no dentist at all, six (50%) responded neutral, with four (33%) preferring teledentistry, and Comments from the medical personnel regarding intimidation by the technology and added workﬂows may surmise why utilization was low. In a busy medical setting, December 2021 \| Volume 2 \| Article 769988 6 Physicians to Dentists via Teledentistry Hammersmith et al. 1. Mostajer Haqiqi A, Bedos C, Macdonald ME. The emergency department as a ’last resort’: why parents seek care for their child’s nontraumatic dental problems in the emergency room. Community Dent Oral Epidemiol. (2016) 44:493–503. doi: 10.1111/cdoe.12239 2. Dickson-Swift V, Kenny A, Gussy M, McCarthy C, Bracksley-O’Grady S. The knowledge and practice of pediatricians in children’s oral health: a REFERENCES problems in the emergency room. Community Dent Oral Epidemiol. (2016) 44:493–503. doi: 10.1111/cdoe.12239 2. Dickson-Swift V, Kenny A, Gussy M, McCarthy C, Bracksley-O’Grady S. 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Frontiers in Oral Health \| www.frontiersin.org REFERENCES doi: 10.1016/j.resuscitation.2018.01.045 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. 14. Marcolino MS, Maia LM, Oliveira JAQ, Melo LDR, Pereira BLD, Andrade- Junior DF, et al. Impact of telemedicine interventions on mortality in patients with acute myocardial infarction: a systematic review and meta-analysis. Heart. (2019) 105:1479–86. doi: 10.1136/heartjnl-2018- 314539 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. 15. Nadar M, Jouvet P, Tucci M, Toledano B, Sicotte C. Impact of synchronous telemedicine models on clinical outcomes in pediatric acute care settings: a systematic review. Pediatr Crit Care Med. (2018) 19:e662–71. doi: 10.1097/PCC.0000000000001733 Copyright © 2021 Hammersmith, Thiel, Messina, Casamassimo and Townsend. 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. 16. Ward MM, Jaana M, Natafgi N. Systematic review of telemedicine applications in emergency rooms. Int J Med Inform. (2015) 84:601–16. doi: 10.1016/j.ijmedinf.2015.05.009 17. Barnett ML, Ray KN, Souza J, Mehrotra A. Trends in telemedicine use in a large commercially insured population, 2005-2017. JAMA. (2018) 320:2147–9. doi: 10.1001/jama.2018.12354 December 2021 \| Volume 2 \| Article 769988 Frontiers in Oral Health \| www.frontiersin.org 8
https://openalex.org/W2025642356	https://europepmc.org/articles/pmc3842255?pdf=render	English	null	Personality Variation in Little Brown Bats	PloS one	2,013	cc-by	7,999	Introduction Phenotypic variation within a species is a requirement for natural selection yet, in animal ecology and behaviour, intra- specific and intra-population variation have often been viewed as more of a nuisance than a biologically important phenomenon [1]. By definition, the study of animal personality, or consistent individual differences in behaviour, challenges this traditional view of variation, and is advancing our understanding of links between behaviour, physiology, and ecology [2]. Five ecologically relevant dimensions of personality have been well defined [2], including reaction to risky situations (i.e., shyness/boldness; e.g., [3,4]), reaction to novel objects or situations (i.e., exploration/avoidance; e.g., [5–7]), activity levels (e.g., [8]), agonistic reactions to conspecifics (i.e., aggressiveness; e.g., [9,10]), and non-aggressive reactions to the presence or absence of conspecifics (i.e., sociability; e.g., [11]). These inter-individual differences in behaviour tend to be repeatable across time, and can be heritable across generations [2,6,12,13]. Recent studies have also shown that personality may impact life history and fitness via relationships to resource acquisition, reaction to predators, reproductive ability, and longevity [8,13,14,15]. Variation in personality may also correlate with metabolism and energetics [16], although the ubiquity of these patterns and directions of relationships within species have not been fully established (e.g., [17]). Although repeatability is fundamental to the definition of personality, ontogenetic or seasonal changes during development or reproductive cycles could cause within-individual variation in personality traits (e.g., activity, exploration propensity, boldness). This temporal variation would not necessarily preclude repeat- ability if between-individual variation at any point during the life cycle exceeded that within individuals or, in other words, the rank of individuals relative to one another remains consistent over time [20]. Especially pronounced shifts in personality traits have been observed at sexual maturity [20,21], but development and reliance on parental care prior to sexual maturation could also influence within-individual variation. For species with precocial offspring, personality traits should remain relatively stable throughout ontogeny, as even young individuals of such species employ adult-like behaviours (e.g., [22,23]). This consistency has been documented for a range of personality traits in precocial species (e.g., docility in ewes, [22]; boldness in deer, [23]; temperament and stress response in cattle, [24]). For altricial species, however, personality traits could change dramatically throughout develop- ment prior to sexual maturity as offspring acquire skills, exhibit more adult-like behaviours, and become self-sufficient (e.g., To be considered personality, individual differences in behav- iour must be repeatable over time and/or across situations [2]. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or pre Funding: Funding was provided by a Natural Sciences and Engineering Research Council (NSERC) Canada Graduate Scholarship to AKM and post-doctoral fellowship to LPM as well as grants to CKRW from NSERC, the Canada Foundation for Innovation, the Manitoba Research and Innovation Fund and Manitoba Hydro Forest Enhancement Program. 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. mpeting Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: allysonmenzies@gmail.com also be thought of as a first step towards determining if a behavioural phenotype is heritable [18,19]. However, repeatability of many traits has not been well quantified for numerous species. For example, many studies have quantified repeatability of mating and courtship behaviours (e.g., mating calls/vocalization, mate choice) and anti-predator behaviour, but fewer have quantified repeatability of other personality traits, such as activity or exploration [12], especially in free-ranging mammals. Personality Variation in Little Brown Bats Allyson K. Menzies*, Mary E. Timonin, Liam P. McGuire, Craig K. R. Willis Department of Biology and Centre for Forest Interdisciplinary Research (C-FIR), University of Winnipeg, Manitoba, Canada Abstract Animal personality or temperament refers to individual differences in behaviour that are repeatable over time and across contexts. Personality has been linked to life-history traits, energetic traits and fitness, with implications for the evolution of behaviour. Personality has been quantified for a range of taxa (e.g., fish, songbirds, small mammals) but, so far, there has been little work on personality in bats, despite their diversity and potential as a model taxon for comparative studies. We used a novel environment test to quantify personality in little brown bats (Myotis lucifugus) and assess the short-term repeatability of a range of behaviours. We tested the hypothesis that development influences values of personality traits and predicted that trait values associated with activity would increase between newly volant, pre-weaning young-of-the- year (YOY) and more mature, self-sufficient YOY. We identified personality dimensions that were consistent with past studies of other taxa and found that these traits were repeatable over a 24-hour period. Consistent with our prediction, older YOY captured at a fall swarming site prior to hibernation had higher activity scores than younger YOY bats captured at a maternity colony, suggesting that personality traits vary as development progresses in YOY bats. Thus, we found evidence of short-term consistency of personality within individuals but with the potential for temporal flexibility of traits, depending on age. Citation: Menzies AK, Timonin ME, McGuire LP, Willis CKR (2013) Personality Variation in Little Brown Bats. PLoS ONE 8(11): e80230. doi:10.1371/ journal.pone.0080230 Received April 25, 2013; Accepted October 1, 2013; Published November 27, nzies et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Menzies et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by a Natural Sciences and Engineering Research Council (NSERC) Canada Graduate Scholarship fellowship to LPM as well as grants to CKRW from NSERC, the Canada Foundation for Innovation, the Manitoba Research and Inno Hydro Forest Enhancement Program. Capture All procedures and fieldwork were conducted under a Manitoba Conservation wildlife scientific permit (permit # WB11145), and were approved by the University of Winnipeg Animal Care Committee. We captured bats using harp traps and mist nets set at the entrance of the hibernaculum and maternity roost. Captured individuals were sexed, weighed to the nearest 0.1 g, and classified as adult or YOY based on the degree of ossification of the third metacarpal-phalangeal joint [37]. We also recorded forearm length and classified adult females as pregnant (presence of a foetus upon gentle abdominal palpitation), lactating (expression of milk and enlarged nipples surrounded by hairless patches) or non- reproductive. We used the residuals of a linear regression between mass and forearm length as a measure of body condition. Our first objective was to test whether a modified version of the hole-board test, a standard open-field behavioural test commonly used to assess personality traits in rodents, would allow us to identify the same personality dimensions in bats as found in other taxa. We also aimed to quantify short-term repeatability of these traits by testing individuals on consecutive nights. We then used these data to test the hypothesis that personality traits may vary at sexual maturity and throughout the reproductive cycle by investigating whether the relative age of YOY or the reproductive status of adults influences variation in personality traits. Little brown bats are ideal for testing these hypotheses since they are an altricial mammal characterized by dramatic changes in the parental dependence of YOY between parturition and the first hibernation season. We predicted that older YOY bats captured during the fall swarming period would be more active and exploratory than younger YOY captured emerging from a maternity colony because YOY bats at mating swarms have reached independence and are preparing for hibernation. We transported bats to a field laboratory 10–40 km from the site of capture, where they were housed for up to 3 days. In all cases, the animals were housed in a dimly lit, quiet room, at room temperature, under natural photoperiod. We housed both summer and fall YOY bats in individual cloth bags (2009) and adult bats in wire-mesh cages (20 cm625 cm621 cm) covered with fabric, in groups of up to 6 individuals (2010). In both years, animals were handled minimally during the brief captivity period. Study Area and Subjects Little brown bats were captured in Manitoba, Canada, during the summers of 2009 and 2010. In early August 2009, newly volant YOY bats (hereafter summer YOY) were caught emerging from a maternity colony in a building in Altamont, MB (49u219N 98u359W). In late August of 2009, older, presumably independent YOY (hereafter fall YOY) were captured during pre-hibernation swarming at Microwave Cave [35], a hibernaculum approximate- ly 50 km north of Grand Rapids, MB (53u129N 99u199W). From June until August of 2010, reproductive and non-reproductive adult bats were captured at maternity colonies in both Altamont and Grand Rapids. The two field sites (Altamont and Grand Rapids) are approximately 400 km apart but bats at these two sites likely belong to the same population and experience similar conditions throughout the year. Based on our long-term banding studies of bats in Manitoba, individual little brown bats in this area routinely travel distances exceeding 500 km in one year [36], and there is very high gene flow among bats, and little evidence of population genetic structure at these sites (Martinez-Nunez and Willis, unpublished). Moreover, given the lack of suitable geology (i.e., limestone karst, abandoned mines) within hundreds of km of Altamont, individuals from the maternity roost there likely swarm at and hibernate in caves near Grand Rapids or other similar hibernacula in central Manitoba. Thus, there is a very high likelihood bats from both capture locations belong to the same population and experience similar conditions throughout their lives. A growing number of species are being studied in the context of personality, but there is still potential to diversify the field considerably. While the model organism approach has obvious value, the features which make a given species an ideal model for behavioural studies (e.g., readily captured or observed repeatedly, tractable in captivity), could also bias our understanding of personality variation [28]. One recent study examined ‘‘behav- ioural types’’ in big brown bats (Eptesicus fuscus; [29]) and bats, in general, have potential as another useful model for personality studies. Although many species will not be ideal for studies of long- term repeatability, as they are difficult to recapture in the wild, other aspects of their biology are highly amenable to comparative studies. Study Area and Subjects For one, with approximately 1200 species, bats represent over 20% of extant mammals and exhibit large ecological and morphological diversity [30], which would allow for studies of personality variation across species in a phylogenetic and environmental context. Moreover, many species can be captured in large numbers, making them suitable for addressing questions requiring large sample sizes. As a result, they represent a potentially valuable taxon for studies of personality variation within species and for comparative studies addressing variation across species, similar to other better-studied taxa (e.g., non- human primates, [31]). We quantified personality of the little brown bat (Myotis lucifugus). This species is one of the most common and widely distributed bat species in North America, although it is suffering high mortality in eastern North America due to the emerging disease white-nose syndrome [32]. Little brown bats hibernate for the winter in mines and caves in groups varying in size from a few to thousands of individuals [33]. In spring, females form maternity colonies where they give birth and care for highly dependent young, until these young-of-the-year (YOY) are capable of foraging on their own [34]. Males disperse individually or in small groups [33]. During the fall, prior to hibernation, large numbers of males, females, and independent YOY congregate in swarms outside hibernacula each night, presumably for mating and other social functions [34]. Capture We provided water via disposable pipette twice daily and fed bats mealworms (Tenebrio molitor) after their personality trials. In total, we captured 76 bats for behavioural testing (n = 28 YOY; n = 20 adult males; n = 28 adult females). Introduction Repeatability represents the upper bound to heritability, and may be easier to measure than genetic relatedness in the field, so it can November 2013 \| Volume 8 \| Issue 11 \| e80230 1 PLOS ONE \| www.plosone.org Personality in Bats Personality in Bats Personality in Bats foraging behaviour in juvenile birds, [25–27]). A better under- standing of within-individual variation in personality traits at different points in the life cycle, particularly for altricial mammals, is important because of the potential impact this variation can have on reproductive fitness and selection on behaviour [20]. Hole-board Test Given that there are no standardized personality tests for bats, we chose to quantify activity and exploratory behaviour using a modified rodent hole-board test (e.g., [38–40]). Little brown bats are highly adept at climbing and crawling and must often crawl to explore cracks, crevices and other potential roost sites, so we reasoned that the hole-board test, with some minor modifications, PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org November 2013 \| Volume 8 \| Issue 11 \| e80230 November 2013 \| Volume 8 \| Issue 11 \| e80230 2 Personality in Bats would provide an ecologically relevant assessment of personality. The testing apparatus (Figure 1) was constructed to hang vertically, and consisted of a test chamber (57642614 cm) with a transparent cover and plastic window screening on the back surface to facilitate climbing by the bats. Four blind holes (2 cm deep by 3 cm diameter) were drilled into this surface. Two holes were positioned closer to the centre of the chamber (15 cm from nearest wall), while the other holes were positioned closer to the corners (5.5 cm from nearest wall). Investigation of the holes closer to the centre of the test is thought to indicate more thorough exploration and lower anxiety. A start box (14.5614.5 cm) was attached to the bottom of the testing chamber, with a sliding door to separate the bat from the test until behavioural recording began. near the corner or the centre of the test, relevant to how bats might search potential roost openings in the wild), latency to head dip (the length of time it took each individual to first explore one of the holes), as well as latency to enter the test (the length of time it took each individual to enter the arena). We also measured the proportion of time spent grooming, as bursts of grooming are indicators of anxiety in rodents [42]. Repeatability Although it was not possible to capture the same individual bats over periods of weeks to months, we conducted two behavioural trials, separated by an interval of approximately 24-hours, to quantify short-term, within-individual repeatability. The second test for each individual was conducted under identical conditions, the night following the first behavioural test, controlling for factors likely to influence differences in behaviour among individuals (e.g., nutritional state assessed via body condition). At the end of the second set of behavioural trials, bats were released at their site of capture. To ensure that recaptured individuals could be identified, each bat was banded using lipped, numbered aluminum bands (Porzana Ltd., 2.9 mm, Icklesham, East Sussex, UK; 2009), or by subcutaneously injecting a passive integrated transponder (PIT) tag (Trovan Ltd. ID 100-01, Douglas, UK; 2010) prior to release. g Little brown bats are prone to enter torpor [41], so prior to behaviour testing we measured each individual’s body tempera- ture by inserting a 1 mm diameter thermocouple probe 3 mm into the rectum. Bats were not tested until body temperature exceeded 30uC. Once normothermic, each bat was placed, individually, in the start box for two minutes before the sliding door was opened. Bats were given a maximum of one minute to enter the test on their own, after which they were gently pushed into the testing chamber with a smooth, plastic plunger. The sliding door was then closed to prevent re-entry. To simulate natural conditions, all trials were run in a darkened room at night, and the behaviour of each bat was recorded for 10 minutes using an infrared video camera (Sony AVCHD Handycam HDR-XR 550) mounted on a tripod. At the end of the trial, individuals were removed from the test and, to eliminate olfactory cues, the test was cleaned using mild, unscented dish detergent and water. All bats were fed mealworms and provided water after testing. November 2013 \| Volume 8 \| Issue 11 \| e80230 Statistical Analyses It is calculated by dividing the variance among individuals (s2 a) by the total variance (where s2 is the variance within individuals over time; [12,18,48]): All PC scores were significantly repeatable from test 1 to test 2 (PC1, ICC = 0.34, d.f. = 73, p = 0.001; PC2, ICC = 0.29, d.f. = 73, p = 0.006; PC3, ICC = 0.25, d.f. = 73, p = 0.02; Figure 2). When we analyzed the individual behaviours that loaded strongly on PC1, PC2 and PC3, frequency of flights (ICC = 0.30, d.f. = 75, p = 0.004), time spent echolocating (ICC = 0.36, d.f. = 75, p = 0.001), latency to enter the test (ICC = 0.38, d.f. = 75, p,0.001), and latency to head dip in the holes closer to the corners of the test (ICC = 0.26, d.f. = 75, p = 0.01) were signifi- cantly repeatable over the 24-hour interval. ICC~sa 2 sa 2zs2 We used ANCOVA to determine effects of age, sex, body size, and body condition on PC scores. We tested the influence of these effects on PC scores from the first trial only, since trial one assessed the response to a novel environment. We did not include sampling location as a factor in our analyses since there is a very high likelihood that bats from both capture locations belong to the same population and experience similar conditions throughout their lives ( [36]; Martinez-Nunez and Willis, unpublished) and because relative age is the difference among the groups of YOY that we assumed would influence personality most strongly. We also omitted sampling year from the analysis because we captured exclusively YOY in 2009 and adults in 2010 so these variables were confounded. We also assumed that the effect of demographic (which has a large influence on most aspects of biology of bats) should be more pronounced. We used backwards, stepwise elimination to remove non-significant interactions and main effects until only significant terms remained (e.g. [8,22,46,49]). To verify our findings we also compared a series of nested models. This analysis provided identical results as the step-wise model reduction so we only report results from the model reduction. We assessed the effect of relative age on PC scores of YOY bats and included sex, forearm length, and body condition as predictor variables. Statistical Analyses All PC scores were significantly repeatable from test 1 to test 2 (PC1, ICC = 0.34, d.f. = 73, p = 0.001; PC2, ICC = 0.29, d.f. = 73, p = 0.006; PC3, ICC = 0.25, d.f. = 73, p = 0.02; Figure 2). When we analyzed the individual behaviours that loaded strongly on PC1, PC2 and PC3, frequency of flights (ICC = 0.30, d.f. = 75, p = 0.004), time spent echolocating (ICC = 0.36, d.f. = 75, p = 0.001), latency to enter the test (ICC = 0.38, d.f. = 75, p,0.001), and latency to head dip in the holes closer to the corners of the test (ICC = 0.26, d.f. = 75, p = 0.01) were signifi- cantly repeatable over the 24-hour interval. PCA. We used the prcomp command in R, which generates PC scores based on singular value decomposition. In order to choose which principal components to retain, we used the Kaiser- Guttman criterion (eigenvalues .1; [44]) and a 10% minimum variance threshold for retention of a given component. To verify our choice of components, we also conducted a parallel analysis [47] using the paran package in R. PC scores from the PCA were used as representative personality scores in subsequent analyses. the first principal component (adjusted eigenvalue = 1.8) and provided weaker support for the second and third (adjusted eigenvalues = 0.8 in both cases). Locomotion, number of flight attempts, and echolocation contributed most strongly to the first component (PC1); individuals with high scores for PC1 had high levels of activity. Behaviours associated with exploration propen- sity contributed to PC2. Individuals with high scores for PC2 exhibited fast, superficial exploration, with shorter latency to enter the arena but longer latencies to head dip in the holes in the corners of the test. The third component (PC3) appeared to reflect anxiety and was represented most strongly by grooming as well as latency to head dip in the holes closer to the center of the arena. We used intra-class correlation (ICC) to test for within- individual repeatability of PC scores and individual behaviours (e.g., [12]). The intra-class correlation coefficient is often used to assess reliability of measurements made by two different observers, or repeatability of traits within individuals, and is based on the mean squares as calculated for a standard ANOVA. Statistical Analyses Results for Principal Component Analysis of behavioural responses of 76 little brown bats in a novel- environment test. Levene’s tests were used to test for equality of variance among groups, Kolmogorov-Smirnov tests were used to test for normality, and data were log transformed as necessary to meet assumptions of parametric analyses. Significance for all tests was assessed at an alpha level of 0.05. Statistical Analyses Forearm length and body condition differed between the sexes (female vespertilionid bats are often larger and in better body condition than males, [50]), thus we assessed the effect of reproductive status, body condition, and forearm length on PC scores of adult female bats, and the effect of forearm length and body condition on male bats, separately. After determining that there was no effect of either forearm length or body condition on either sex, we pooled females and males to determine the effect of sex on personality scores in all adult bats. Fall YOY had significantly higher activity scores (PC1) than summer YOY (F1,25 = 44.7, p,0.001; Figure 3), with no effect of sex (F1,23 = 0.3, p = 0.59), forearm length (F1,19 = 0.3, p = 0.58), or body condition (F1,18 = 0.01, p = 0.98). Fall YOY exhibited significantly higher levels of all measures of activity during trial 1 (locomotion, t = 6.2, d.f. = 25, p,0.001; echolocation, t = 3.5, d.f. = 25, p,0.01; flights, t = 4.3, d.f. = 25, p,0.001). None of the factors in our models had a significant effect on PC2 (age, F1,25 = 2.1, p = 0.15; sex, F1,17 = 0.1, p = 0.72; forearm, F1,19 = 0.84, p = 0.37; body condition, F1,21 = 0.2, p = 0.70) or PC3 (age, F1,25 = 2.1, p = 0.16; sex, F1,19 = 0.1, p = 0.74; forearm, F1,18 = 0.8, p = 0.40; body condition, F1,21 = 0.2, p = 0.70) for YOY bats. Table 1. Results for Principal Component Analysis of behavioural responses of 76 little brown bats in a novel- environment test. Variables Components 1 2 3 Locomotion 0.481 20.324 0.042 Echolocation 0.422 0.087 20.096 Flight 0.455 0.171 20.040 Latency to Enter 20.339 0.556 20.159 Latency to Head Dip in Holes (edge) 20.237 20.714 20.092 Latency to Head Dip in Holes (centre) 20.382 20.192 0.412 Grooming 20.257 0.052 20.886 Standard Deviation 1.47 1.03 0.97 % of total variance 31.4 15.4 13.6 Cumulative Variance (%) 31.4 46.7 60.4 Principal Components retained met the Kaiser-Guttman criterion (i.e., eigenvalues .1, [44]). Bolded eigenvectors represent factors with loadings .0.4, which were considered to have contributed significantly to a particular component [40]. doi:10.1371/journal.pone.0080230.t001 Table 1. Results for Principal Component Analysis of behavioural responses of 76 little brown bats in a novel- environment test. Table 1. Statistical Analyses All statistical analyses were conducted in R version 2.15.0 GUI 1.51 [43]. Principal Component Analysis (PCA) was used to reduce the number of measured behavioural traits into composite, synthetic variables or principal component (PC) scores (e.g., [17,40]). An alternative method is Regularized Exploratory Factor Analysis (REFA), a data reduction method designed for small sample sizes. However, our dataset is well-suited to PCA (sample size larger than 50 individuals and relatively few variables included, [45]) so, for these reasons and to be consistent with previous studies addressing personality variation in wild-captured vertebrates (e.g., [8,17,40,46]), we used PCA. All variables were scaled and centered (by subtracting the mean from each value and dividing this by the standard deviation) prior to inclusion in the Videos were scored for a range of behaviours we selected based on previous studies of rodents (e.g., [39,40]), their ecological relevance to bats, and our preliminary observations of bats in the hole-board test. We scored locomotion (proportion of time spent crawling or climbing in the test), frequency of flight attempts, the proportion of time spent echolocating (i.e., when the bats scanned the arena with their mouths open), frequency of head dips (the number of times an individual explored one of the holes either Figure 1. Screen capture of our modified hole-board test from a video recording of a behaviour trial. doi:10.1371/journal.pone.0080230.g001 Figure 1. Screen capture of our modified hole-board test from a video recording of a behaviour trial. doi:10.1371/journal.pone.0080230.g001 November 2013 \| Volume 8 \| Issue 11 \| e80230 November 2013 \| Volume 8 \| Issue 11 \| e80230 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 Personality in Bats the first principal component (adjusted eigenvalue = 1.8) and provided weaker support for the second and third (adjusted eigenvalues = 0.8 in both cases). Locomotion, number of flight attempts, and echolocation contributed most strongly to the first component (PC1); individuals with high scores for PC1 had high levels of activity. Behaviours associated with exploration propen- sity contributed to PC2. Individuals with high scores for PC2 exhibited fast, superficial exploration, with shorter latency to enter the arena but longer latencies to head dip in the holes in the corners of the test. The third component (PC3) appeared to reflect anxiety and was represented most strongly by grooming as well as latency to head dip in the holes closer to the center of the arena. Discussion Based on our novel environment test, we found evidence for personality traits in little brown bats similar to those previously identified in rodents (e.g., [7,40]), songbirds (e.g., [5,6]) and fish (e.g., [51,52]). In particular, behaviours reflecting activity, including locomotion, flight, and echolocation, separated into one component (PC1) while the second component (PC2) established a relationship between latency to enter the test and latency to head dip in holes closest to the walls of the test (i.e., holes 3 and 4). The hole-board test was initially developed to help separate activity components of personality in rodents from exploration components, which the standard novel environment test (i.e., without holes to investigate) failed to do [39]. We found that behaviours associated with exploration and activity separated into distinct components, suggesting that this test also isolates these personality dimensions in bats. The final two behaviours, grooming and latency to head dip in holes closer to the centre of the test (i.e., holes 1 and 2) grouped onto PC3. Although this link is unknown for bats, in rodents, bursts of grooming behaviour are a component of the stress response and indicate increased anxiety [42]. Latency to head dip in the holes that are more exposed (i.e., closer to the center of the arena) may also reflect anxiety as longer latencies for exploring these holes could indicate apprehension to venture away from the walls of the test. It would be useful to better define this personality dimension and assess whether these behaviours are associated with physiological correlates of stress (e.g., levels of circulating glucocorticoid hormones) in bats. These three components all satisfied the Kaiser-Guttman criterion, the objective criterion used in most other studies to retain or reject or reject personality dimensions (e.g., [8,17,40,46]), as well as explaining a minimum of 10% of the total variation in our dataset. Strictly speaking, PC2 and PC3 fell just below the criterion for inclusion based on a parallel analysis Figure 2. Scatterplots demonstrating repeatability of (a) PC1, (b) PC2 and (c) PC3 between behavioural trials 1 and 2 for 76 little brown bats in a novel environment test. Note that reduced- major-axis regression lines are plotted to illustrate the relationship, but repeatability was assessed using an intra-class correlation. Results Three principal components each explained a minimum of 10% of the variance in recorded behaviours, met the Kaiser-Guttman criterion, and, cumulatively, explained over 60% of the total variance in the dataset (Table 1). The parallel analysis supported November 2013 \| Volume 8 \| Issue 11 \| e80230 PLOS ONE \| www.plosone.org 4 Personality in Bats Reproductive status did not have an effect on any perso Figure 2. Scatterplots demonstrating repeatability of (a) (b) PC2 and (c) PC3 between behavioural trials 1 and 2 f little brown bats in a novel environment test. Note that red major-axis regression lines are plotted to illustrate the relationshi repeatability was assessed using an intra-class correlation. doi:10.1371/journal.pone.0080230.g002 Figure 3. Comparison of activity levels for 28 YOY little brown bats captured at a maternity colony (summer YOY, n = 9) and a hibernaculum (fall YOY, n = 18) in August of 2009. doi:10.1371/journal.pone.0080230.g003 Figure 3. Comparison of activity levels for 28 YOY little brown bats captured at a maternity colony (summer YOY, n = 9) and a hibernaculum (fall YOY, n = 18) in August of 2009. doi:10.1371/journal.pone.0080230.g003 F1,25 = 0.3, p = 0.61; body condition, F1,26 = 0.9, p = 0.35) or PC3 (forearm, F1,26 = 3.4, p = 0.08; body condition, F1,25 = 0.01, p = 0.92). Similarly, in adult males, there was no effect of body condition or forearm length on PC1 (forearm, F1,18 = 1.5, p = 0.24; body condition, F1,17 = 1.2, p = 0.29), PC2 (forearm, F1,18 = 0.8, p = 0.37; body condition, F1,17 = 0.9, p = 0.37) or PC3 (forearm, F1,17 = 2.0, p = 0.18; body condition, F1,18 = 0.9, p = 0.36). Finally, there was no effect of sex on PC1 (F1,41 = 2.5, p = 0.12), PC2 (F1,41 = 1.2, p = 0.29) or PC3 (F1,43 = 3.1, p = 0.09) of adult bats. Figure 3. Comparison of activity levels for 28 YOY little brown bats captured at a maternity colony (summer YOY, n = 9) and a hibernaculum (fall YOY, n = 18) in August of 2009. doi:10.1371/journal.pone.0080230.g003 F1,25 = 0.3, p = 0.61; body condition, F1,26 = 0.9, p = 0.35) or PC3 (forearm, F1,26 = 3.4, p = 0.08; body condition, F1,25 = 0.01, p = 0.92). Results Similarly, in adult males, there was no effect of body condition or forearm length on PC1 (forearm, F1,18 = 1.5, p = 0.24; body condition, F1,17 = 1.2, p = 0.29), PC2 (forearm, F1,18 = 0.8, p = 0.37; body condition, F1,17 = 0.9, p = 0.37) or PC3 (forearm, F1,17 = 2.0, p = 0.18; body condition, F1,18 = 0.9, p = 0.36). Finally, there was no effect of sex on PC1 (F1,41 = 2.5, p = 0.12), PC2 (F1,41 = 1.2, p = 0.29) or PC3 (F1,43 = 3.1, p = 0.09) of adult bats. Discussion For some bat species at some roosts it will be possible to recapture sufficient numbers of individual bats across longer intervals because individuals show high fidelity to specific sites throughout their lives (e.g., [36]). Holding bats in captivity for longer periods could also allow for longer intervals between tests although captivity could influence behaviour. We recommend both approaches for future studies. Another possibility is that the difference between summer and fall YOY reflects links between life history, ecology and personality. The ‘‘fast’’, highly active individuals may have also been quick to reach independence and disperse to swarming sites while the ‘‘slower’’, less active individuals may have taken longer to become independent, and remained at the maternity roosts for longer. Previous studies have demonstrated that activity in novel environments is correlated with dispersal in the field (e.g., Parus major, [58]) and that dispersal latency decreases with increasing exploratory activity (e.g., Mus musculus musculus, [59]), which is consistent with our results. More active YOY may have dispersed from the natal roost and ventured to swarming sites earlier. Taken together, these findings highlight an important consideration for studies of personality that address behavioural differences of different age classes of animals. Grouping all YOY bats together, regardless of developmental stage, would have masked subtle but potentially important differences in behaviour within this age class. We found evidence that personality traits in bats differentiate into similar components as those described in past studies of rodents, fish, and birds, and that the open-field component of the hole-board test was useful for measuring activity and exploration. Other standardized behavioural tests are needed to validate results found in this study and to further examine a range of personality traits in this taxon. We also found evidence of temporal flexibility in activity of YOY bats. Although most studies control for the age of subjects, typically all YOY are considered as one discrete group. Our results indicate that important biological differences exist within this category. Since within-individual variation of person- ality traits in bats has received relatively little attention, further studies are necessary to understand both the consistency and temporal flexibility of personality in bats. Future studies investi- gating consistency, or inconsistency, of personality traits through- out energetically demanding portions of the life cycles (i.e., mating/reproduction, preparing for hibernation) for both adult male and adult female bats may also help shed light on potential links between personality and energetics. Discussion doi:10.1371/journal.pone.0080230.g002 Reproductive status did not have an effect on any personality scores in adult females (PC1, F2,22 = 0.4, p = 0.67; PC2, F2,22 = 0.5, p = 0.59; PC3, F2,22 = 0.5, p = 0.62). There was also no effect of forearm length or body condition on PC1 (forearm, F1,26 = 4.0, p = 0.06; body condition, F1,25 = 3.6, p = 0.15), PC2 (forearm, November 2013 \| Volume 8 \| Issue 11 \| e80230 PLOS ONE \| www.plosone.org 5 Personality in Bats (adjusted eigenvalue = 0.8), but given that they satisfied criteria used in other comparable studies and their clear biological significance we analyzed all three in more detail. rely on echolocation for foraging and navigation (e.g., [56,57]). These results support our prediction that YOY would show increased activity in conjunction with this critical shift in development. Individual bats reacted similarly during repeated tests and scores for all three principal components and were significantly repeatable over a 24-hour period (Figure 2). The intra-class correlation coefficients that we obtained (0.25–0.35) were compa- rable but slightly below the mean (r = 0.37) for published estimates for a wide range of behavioural traits in 114 species (reviewed by [12]). Our 24-hour inter-test interval fell within the range of published inter-test intervals for quantifying repeatability of behaviours in vertebrates (e.g., [53,54]) but it was also relatively short. One potential limitation of novel environment tests repeated at short inter-test intervals is that some individuals may quickly habituate and change their behaviour between tests, while others might behave more consistently across trials. This individual variation in habituation behaviour reduces the potential to detect repeatability [55]. Based on our experience with bats, some individuals readily adapt to captivity and learn to eat novel food within 24 hours of capture, while others take significantly longer. As a result, some of the bats we tested may have habituated to the test quickly and responded differently in the second test causing us to underestimate trait repeatability [18]. In general, habituation is less likely over a longer inter-test time interval (e.g., a month or a year) compared to a shorter interval, reducing the within- individual variation between trials. Ideally, repeatability should be assessed over longer periods of weeks, months, or even years for long-lived species. Recapture rates are typically low for free- ranging bats and we were not able to recapture individuals in this study. Acknowledgments We thank C. Carrie`re and all other members of the University of Winnipeg Bat Lab for help with fieldwork. We are grateful to S. Forbes, J. Franck, M. Wiegand, A. Weiss and an anonymous reviewer for helpful comments on earlier versions of this manuscript. We also thank the residents of Altamont, MB and Misipawistik First Nation for access to the study area and Manitoba Conservation for logistical support and lodging in the field. Discussion We found evidence supporting the hypothesis that age of YOY influences variation in personality of wild-captured bats. Consis- tent with our predictions, fall YOY displayed elevated activity levels in conjunction with a critical shift in their independence and maturity (Figure 3). In a species that relies on flight, this shift could reflect morphological development (e.g., of the wings and pectoral muscles, which affect flight ability, [26]). However, we found no difference in body mass, forearm length or body condition between the two groups which indicates that volant YOY from both groups had reached adult size when we assessed their behaviour. Thus, higher activity levels of fall YOY could reflect an ontogenetic shift in behaviour as these bats became independent. Summer YOY were captured at the natal roost, and were likely still reliant on their mothers and not yet self-sufficient. In contrast, fall YOY were presumably independent, as they had already left their maternity colony and traveled to a hibernaculum (which can be 30–500 km from the natal roost in our study area; [36]). Fall YOY would also need to have more developed echolocation, one of the measures of activity that contributed to PC1, because bats Author Contributions Conceived and designed the experiments: AKM MET CKRW. Performed the experiments: AKM MET. Analyzed the data: AKM MET LPM CKRW. Wrote the paper: AKM CKRW. Conceived and designed the experiments: AKM MET CKRW. Performed the experiments: AKM MET. Analyzed the data: AKM MET LPM CKRW. Wrote the paper: AKM CKRW. 5. Verbeek MEM, Drent PJ, Wiepkema PR (1993) Consistent individual differences in early exploratory behaviour of male great tits. Anim Behav 48: 1113–1121. 4. 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Dingemanse NJ, Both C, Drent PJ, Van Oers K, Van Noordwijk AJ (2002) Repeatability and heritability of exploratory behaviour in great tits from the wild. Anim Behav 64: 929–938. 33. Fenton MB, Barclay RMR (1980) Myotis lucifugus. Mammalian Species 142: 1–8. 34. Fenton MB (1969) Summer activity of Myotis lucifugus (Chiroptera: Vespertilio- nidae) at hibernacula in Ontario and Quebec. Can J Zool 47: 597–602. 7. Careau V, Bininda-Emonds ORP, Thomas DW, Re´ale D, Humphries MM (2009) Exploration strategies map along fast-slow metabolic and life history continua in muroid rodents. Funct Ecol 23: 150–156. 35. McRitchie WD, Monson KM (2000) Caves & karst in Manitoba’s Interlake region from surveys conducted by the Speleological Society of Manitoba 2nd Edition. Winnipeg: Speleological Society of Manitoba. 73. Edition. Winnipeg: Speleological Society of Manitoba. 73. continua in muroid rodents. Funct Ecol 23: 150–156. 36. Norquay KJO, Martinez-Nunez F, Dubois JE, Monson KM, Willis CKR (2013) Long-distance movements of little brown bats (Myotis lucifugus). J Mammal 94: 506–515. 8. Boon AK, Re´ale D, Boutin S (2008) Personality, habitat use, and their consequences for survival in North American red squirrels Tamiasciurus hudsonicus. Oikos 117: 1321–1328. 9. Lahti K, Huuskonen H, Laurila A, Piironen J (2002) Metabolic rate and aggressiveness between Brown Trout populations. Funct Ecol 16: 167–174. 37. Anthony EL (1988) Age determination in bats. In: Kunz TH editor. Ecological and behavioural methods for the study of bats. Washington: Smithsonian Institution. 47–58. 10. Bell AM (2005) Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus). J Evolution Biol 18: 464–473. 38. Nolan NA, Parkes MW (1973) The effects of benzodiazepines on the behaviour of mice on a hole-board. Psychopharmacology 29: 277–288. of stickleback (Gasterosteus aculeatus). J Evolution Biol 18: 464–473. 11. Favre M, Martin JGA, Festa-Bianchet M (2008) Determinants and life-history consequences of social dominance in bighorn ewes. Anim Behav 76: 1373–1380. y p gy 39. File SE, Wardill AG (1975) Validity of head-dipping as a measure of exploration in a modified hole-board. Psychopharmacology 44: 53–59. 39. File SE, Wardill AG (1975) Validity of head-dipping as a me 12. Bell AM, Hankison SJ, Laskowski KL (2009) The repeatability of behaviour: a meta-analysis. Anim Behav 77: 771–783. in a modified hole-board. Psychopharmacology 44: 53–59. 40. References Dingemanse NJ, Bouwman KM, van de Pol M, van Overveld T, Patrick SC, et al. (2012) Variation in personality and behavioural plasticity across four populations of the great tit Parus major. J Anim Ecol 81: 116–126. 29. Kilgour RJ, Brigham RM (2013) The relationships between behavioural categories and social influences in the gregarious big brown bat (Eptesicus fuscus). Ethology 119: 1–10. 56. Gould E, Cooley B, Barnick P (1981) Echolocation by young bats on their initial and subsequent flights. Dev Psychobiol 14: 41–54. gy 30. Simmons NB, Conway TM (2003) Evolution of ecological diversity in bats. In: Kunz TH, Fenton MB, editors. Bat ecology. Chicago: The University of Chicago Press. 493–535. 57. de Fanis E, Jones G (1995) Post-natal growth, mother-infant interactions and development of vocalizations in the vespertilionid bat Plecotus auritus. J Zool 235: 85–97. 31. Freeman HD, Gosling SD (2010) Personality in nonhuman primates: a review and evaluation of past research. Am J Primatol 71: 1–19. 58. Dingemanse NJ, Both C, van Noordwijk AJ, Rutten AL, Drent PJ (2003) Natal dispersal and personalities in great tits (Parus major). P R Soc B 270: 741–747. 32. Frick WF, Pollock JF, Hicks AC, Langwig KE, Reynolds DS, et al. (2010) An emerging disease causes regional population collapse of a common North American bat species. Science 329: 679–682. 59. Krackow S (2003) Motivational and heritable determinants of dispersal latency in wild male house mice (Mus musculus musculus). Ethology 109: 671–689. November 2013 \| Volume 8 \| Issue 11 \| e80230 PLOS ONE \| www.plosone.org 7 7
https://openalex.org/W3092821719	https://www.matec-conferences.org/articles/matecconf/pdf/2020/17/matecconf_ti2019_12019.pdf	English	null	Effect of microstructure and cooling rate on the fatigue performance of TIMETAL<sup>®</sup> 575	MATEC web of conferences	2,020	cc-by	5,111	bUniversity of Hertfordshire, Hatfield AL10 9AB bUniversity of Hertfordshire, Hatfield AL10 9AB 1. Introduction Cooling rate has a substantial impact on the microstructure of titanium alloys, altering the morphology and phase composition hence influencing properties. Continuous cooling transformation (CCT) phase diagrams are used to predict cooling rates influence on microstructures and aid in tailoring mechanical properties for a given application. For new development alloys such as Ti575, there is currently no literature or data available to construct a CCT diagram. This work aims to identify some key boundaries on a basic CCT diagram for Ti575 to determine temperatures at which microstructural features begin to form and ultimately be able to optimise processing parameters. Alloys with higher β-phase stability coefficients result in transformations occurring at lower temperatures, and this leads to less diffusion of alloying elements to their respective phases. As a result, less diffusion of alpha stabilisers to secondary alpha (αs) takes place during the β→α+β transformation, producing a finer transformation product. Since more time is required to achieve the same microstructure, the transformation curve will be pushed to the right. As Ti575 has a higher β-phase stability coefficient than Ti-6Al-4V it is predicted that the boundaries identified by the CCT diagram will be further to the right. In near alpha and alpha+beta titanium alloys, large regions of local texture, referred to as “macrozones”, are capable or reducing the fatigue life of components when the fatigue cycle contains a dwell period at peak stress [5]. The local texture can be influenced by both morphological features of the bi-modal microstructure which are utilised for their good balance in fatigue and creep properties. The texture of αp evolves from coarse lamellae that undergoes dynamic spherodisation and recrystallisation mechanisms during thermal mechanical processing, while the texture of αs is inherited from the high temperature β-phase through the Burgers orientation relation and variant selection occurring in the β → α transformation. αs have been known to have favourable variants which align their c-axis with those of neighbouring αp [4] [5]. In this work, EBSD was undertaken to investigate the effect of αp volume fraction and cooling rate on prior beta grain size and αs colony size. Table 1. Nominal composition of Ti575 (wt%) Fe V Si Al O Ti 0.25 7.7 0.5 5.3 0.18 Bal Table 2. Material data Alloy Density (g/cm3) Beta Transus Ti575 4.55 967.5 5ºC 2. Experimental Procedure Table 1. Nominal composition of Ti575 (wt%) Abstract A key design consideration for material selection in the aerospace industry is weight reduction; with excellent strength to weight ratio, high temperature resistance, and fatigue performance, titanium alloys are extensively used. New titanium alloys continue to enhance performance and broaden the range of applications. Titanium Metals Corporation (TIMET) recently developed TIMETAL® 575 (Ti575), a high strength titanium alloy with superior fatigue performance over Ti-6Al-4V, aimed at aerospace applications where these properties are imperative i.e. aerospace turbine discs and blades. [1] [2] This work intends to advance the understanding of the effect of thermal processing of Ti575, by investigating the effect of primary alpha (αp) volume fraction and cooling rate on tensile and fatigue performance in post forged heat-treated microstructures. Microstructural assessment and mechanical performance were completed and are discussed. Three cooling methods from three solution heat-treat temperatures were investigated in this work. The results from these experiments were compared using optical microscopy, electron backscatter diffraction (EBSD), room temperature tensile and high cycle fatigue (HCF) tests. MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032112019 aTIMET UK, PO Box 704. Witton, Birmingham, UK. B72 1LT aTIMET UK, PO Box 704. Witton, Birmingham, UK. B72 1LT © 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. Experimental Procedure Material used in this experiment was sourced from a full-scale triple melted vacuum arc remelted (VAR) ingot of Ti575 manufactured by TIMET UK Witton plant. The ingot received a proprietary sequence of forging operations leading to a 250mm diameter billet. Samples were sectioned from the forged billet into 20x20x120mm test pieces. Samples were held at three different solution heat treatment temperatures (880°C, 910°C and 940°C) for 1 hour to provide varying proportions of αp, and were then cooled using three different cooling mediums (vermiculite, air and oil). Depending on the solution heat treatment temperature and cooling medium, cooling rates varied from 2 to 16°C/s. Cooling curves were measured using an embedded K-type thermocouple located in the centre of a test block which recieved each of the nine different cooling conditions. An example of the thermocouple traces from solution heat treatment at 940°C is shown in Figure 1. © 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/). MATEC Web of Conferences 321, 12019 (2020) MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032112019 The 14th World Conference on Titanium Figure 1. Thermocouple trace for different cooling mediums when cooling from 940°C The end of the dual phase field was identified as the temperature at which the cooling curve resumed a steady rate after the exothermic phase transformation when the test block was air cooled from 1065°C. End of dual phase field is indicated on Figure 2 and Figure 3, established at 737°C. Cooling rate was calculated by the difference in solution heat treat temperature and the end of dual phase field (Tf β → α+β) divided by time. The end of the dual phase field temperature varies with cooling rate since increased undercooling leads to the transformation temperature being suppressed. Due to experimental limitations this was assumed to be a fixed temperature when calculating cooling rates. Figure 2. Thermocouple trace for air cooled Ti575 from 1065°C Figure 3. Cooling rate curve for air cooling Ti575 from 1065°C using 7pt Quadratic Differentiation All samples received an aging treatment at 500°C for 8 hours before mechanical testing was completed. f Figure 1. 2. Experimental Procedure Thermocouple trace for different cooling mediums when cooling from 940°C The end of the dual phase field was identified as the temperature at which the cooling curve resumed a steady rate after the exothermic phase transformation when the test block was air cooled from 1065°C. End of dual phase field is indicated on Figure 2 and Figure 3, established at 737°C. Cooling rate was calculated by the difference in solution heat treat temperature and the end of dual phase field (Tf β → α+β) divided by time. The end of the dual phase field temperature varies with cooling rate since increased undercooling leads to the transformation t t b i d D t i t l li it ti thi d t b fi d t t h l l ti li t Figure 1. Thermocouple trace for different cooling mediums when cooling from 940°C Figure 1. Thermocouple trace for different cooling mediums when cooling from 940°C The end of the dual phase field was identified as the temperature at which the cooling curve resumed a steady rate after the exothermic phase transformation when the test block was air cooled from 1065°C. End of dual phase field is indicated on Figure 2 and Figure 3, established at 737°C. Cooling rate was calculated by the difference in solution heat treat temperature and the end of dual phase field (Tf β → α+β) divided by time. The end of the dual phase field temperature varies with cooling rate since increased undercooling leads to the transformation temperature being suppressed. Due to experimental limitations this was assumed to be a fixed temperature when calculating cooling rates. Figure 1. Thermocouple trace for different cooling mediums when cooling from 940°C The end of the dual phase field was identified as the temperature at which the cooling curve resumed a steady rate after the exothermic phase transformation when the test block was air cooled from 1065°C. End of dual phase field is indicated on Figure 2 and Figure 3, established at 737°C. Cooling rate was calculated by the difference in solution heat treat temperature and the end of dual phase field (Tf β → α+β) divided by time. The end of the dual phase field temperature varies with cooling rate since increased undercooling leads to the transformation temperature being suppressed. Due to experimental limitations this was assumed to be a fixed temperature when calculating cooling rates. MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium HCF were completed with ~100Hz sine wave on a vibrophore under load control and R ≈ 0. Tests were to be terminated if the test piece reached 2 x 107 cycles designated as the “run-out” condition however; no samples survived to run-out. All tests were carried out in accordance with BS EN 6072:2010 at a fixed peak stress of 700MPa. [2] To establish when key microstructure features begin to form, 11 samples sectioned into 20x20x40mm test pieces were heated in the beta phase (1065°C) for 1 hour then air cooled to the temperatures listed in Table 3. Once the temperature in Table 3 was met, the samples were water quenched to “freeze in” the microstructural features at that temperature. The microstructure from each condition was then characterised to establish boundaries on a basic CCT diagram. Temperatures in Table 3 were determined using a test block with embedded thermocouple as before. Table 3. Air cooled temperatures interrupted with water quench Start temperature 1065°C Temperature (°C) Air Cool Time (secs) 1000 12 970 15 940 19 910 23 880 27 850 32 820 38 790 44 760 58 730 72 700 84 Table 3. Air cooled temperatures interrupted with water quench Start temperature 1065°C Temperature (°C) Air Cool Time (secs) 1000 12 970 15 940 19 910 23 880 27 850 32 820 38 790 44 760 58 730 72 700 84 High resolution EBSD maps ~1 x 0.75mm with a 0.2µm step size were generated at TIMET Witton on an FEI Quanta FEG250 equipped with an HKL Channel 5 system. EBSD maps were generated on all samples cooled from 940°C along with samples air cooled from 910°C and 880°C. Table 3. Air cooled temperatures interrupted with water quench High resolution EBSD maps ~1 x 0.75mm with a 0.2µm step size were generated at TIMET Witton on an FEI Quanta FEG250 equipped with an HKL Channel 5 system. EBSD maps were generated on all samples cooled from 940°C along with samples air cooled from 910°C and 880°C. High resolution EBSD maps ~1 x 0.75mm with a 0.2µm step size were generated at TIMET Witton on an FEI Quanta FEG250 equipped with an HKL Channel 5 system. EBSD maps were generated on all samples cooled from 940°C along with samples air cooled from 910°C and 880°C. 2. Experimental Procedure The end of the dual phase field was identified as the temperature at which the cooling curve resumed a steady rate after the exothermic phase transformation when the test block was air cooled from 1065°C. End of dual phase field is indicated on Figure 2 and Figure 3, established at 737°C. Cooling rate was calculated by the difference in solution heat treat temperature and the end of dual phase field (Tf β → α+β) divided by time. The end of the dual phase field temperature varies with cooling rate since increased undercooling leads to the transformation temperature being suppressed. Due to experimental limitations this was assumed to be a fixed temperature when calculating cooling rates. being suppressed. Due to experimental limitations this was assumed to be a fixed temperature when calculating cooling rates. Figure 2. Thermocouple trace for air cooled Ti575 from 1065°C Figure 3. Cooling rate curve for air cooling Ti575 from 1065°C using 7pt Quadratic Differentiation eceived an aging treatment at 500°C for 8 hours before mechanical testing was completed. ated samples were machined into test pieces for tensile and HCF tests, which were carried out at TIMET Witton and Swansea University. Figure 2 Thermocouple trace for air cooled Ti575 from 1065°C Figure 2. Thermocouple trace for air cooled Ti575 from 1065°C Figure 3. Cooling rate curve for air cooling Ti575 from 1065°C using 7pt Quadratic Differentiation Figure 3. Cooling rate curve for air cooling Ti575 from 1065°C using 7pt Quadratic Differentiation All samples received an aging treatment at 500°C for 8 hours before mechanical testing was completed. ere completed under strain control using a strain rate of 0.003s-1 up to yield followed by displacement control using a strain rate of ≤ 0.1s-1 on to fracture. Dual rate tensile tests were completed under strain control using a strain rate of 0.003s-1 up to yield followed by displacement control using a strain rate of ≤ 0.1s-1 on to fracture. 2 https://doi.org/10.1051/matecconf/202032112019 MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium Samples air cooled then water quenched at 30°C intervals are shown on the basic CCT diagram in Figure 5. Within the temperature range of 1000-940°C, a fully martensitic microstructure is formed with no grain boundary alpha. Between 910-880°C, non-continuous alpha begins to form on the prior beta grain boundary. This alpha continues to grow forming continuous grain boundary alpha at 850°C. At 820°C, αs begins to nucleate and grow off the grain boundary alpha as colonies of αs. By 790°C, there are increased amounts of αs growing from the grain boundary along with some αs beginning to form within the beta grain in a basketweave morphology. Both αs colonies from the grain boundary and αs within beta grains form at 760°C. At 730°C the αs lamellae have begun to coarsen and by 700°C aligned alpha colonies have formed. Samples air cooled then water quenched at 30°C intervals are shown on the basic CCT diagram in Figure 5. Within the temperature range of 1000-940°C, a fully martensitic microstructure is formed with no grain boundary alpha. Between 910-880°C, non-continuous alpha begins to form on the prior beta grain boundary. This alpha continues to grow forming continuous grain boundary alpha at 850°C. At 820°C, αs begins to nucleate and grow off the grain boundary alpha as colonies of αs. By 790°C, there are increased amounts of αs growing from the grain boundary along with some αs beginning to form within the beta grain in a basketweave morphology. Both αs colonies from the grain boundary and αs within beta grains form at 760°C. At 730°C the αs lamellae have begun to coarsen and by 700°C aligned alpha colonies have formed. Figure 5. Ti575 air cooled from 1065°C interrupted at 30°C intervals with water quench Figure 5. Ti575 air cooled from 1065°C interrupted at 30°C intervals with water quench Figure 5. Ti575 air cooled from 1065°C interrupted at 30°C intervals with water quench Tensile Tensile Table 4. Tensile results in the solution heat treated and aged condition Table 4. Tensile results in the solution heat treated and aged condition Table 4. Tensile results in the solution heat treated and aged condition MPa % Sample ID 0.2% P.S. U.T.S Elongn. on 5.65√A Elongn. on 4D R. in A. Microstructure MATEC Web of Conferences 321, 12019 (2020) https://doi.org/10.1051/matecconf/202032112019 The 14th World Conference on Titanium MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032112019 Microstructure Since all non-interrupted cooling rates experiments were carried out in the dual phase field, bimodal microstructures were observed with varying proportions of αp based on the solution heat treat temperature. Cooling Ti575 at the rate of 8.37°C/s or faster resulted in the development of a martensitic microstructure seen in Figure 4a, made up of αp and α’ with no grain boundary alpha forming. The formation of martensite was confirmed using high magnification backscattered imaging seen in Figure 4d. For cooling rates between 5.12 and 4.77°C/s, some diffusional transformations begin to take place and a basketweave morphology is observed along with grain boundary alpha situated on prior beta grain boundaries, as represented in Figure 4b. During slower cooling rates, between 2.10 and 3.22°C/s, diffusion controlled nucleation and growth of stable α and β phases in colonies of aligned α-phase lamellae within prior β-phase grains was observed. Significant amounts of grain boundary alpha formation can also be seen, Figure 4c. [3] Figure 4. Optical micrograph of Ti575 SHT at 880°C with a) oil quenched (10.94°C/s cooling rate) b) air-cooled (4.90°C/s cooling rate) c) vermiculite cooled (3.22°C/s cooling rate) d) Backscattered SEM image of martensite formed. Figure 4. Optical micrograph of Ti575 SHT at 880°C with a) oil quenched (10.94°C/s cooling rate) b) air-cooled (4.90°C/s cooling rate) c) vermiculite cooled (3.22°C/s cooling rate) d) Backscattered SEM image of martensite formed. 0°C with a) oil quenched (10.94°C/s cooling rate) b) air-cooled (4.90°C/s cooling rate) c) vermiculite cooled (3.22°C/s cooling rate) d) Backscattered SEM image of martensite formed. 3 Samples air cooled then water quenched at 30°C intervals are shown on the basic CCT diagram in Figure 5. Within the temperature range of 1000-940°C, a fully martensitic microstructure is formed with no grain boundary alpha. Between 910-880°C, non-continuous alpha begins to form on the prior beta grain boundary. This alpha continues to grow forming continuous grain boundary alpha at 850°C. At 820°C, αs begins to nucleate and grow off the grain boundary alpha as colonies of αs. By 790°C, there are increased amounts of αs growing from the grain boundary along with some αs beginning to form within the beta grain in a basketweave morphology. Both αs colonies from the grain boundary and αs within beta grains form at 760°C. At 730°C the αs lamellae have begun to coarsen and by 700°C aligned alpha colonies have formed. Variability can be seen in the calculated cooling rates in Table 4, these are due to the differing temperature range used in the calculation and the sensitivity of the equipment to measure change in temperature at faster cooling rates. MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium Cooling rate (°C/s) 940°C OQ 1305 1447 5.0 5.5 * 15.69 940°C AC 1049 1211 14.5 16.5 31.0 5.12 940°C VC 975 1146 14.5 17.0 36.5 2.10 910°C OQ 1337 1468 8.5 9.5 27.0 8.37 910°C AC 1030 1176 17.0 18.5 46.0 4.77 910°C VC 991 1128 15.5 18.0 39.0 2.66 880°C OQ 1336 1455 11.0 12.5 34.0 10.94 880°C AC 1057 1182 16.5 19.5 46.0 4.90 880°C VC 1018 1129 16.5 19.0 43.5 3.22 *Unable to measure RinA due to a section missing post fracture Variability can be seen in the calculated cooling rates in Table 4, these are due to the differing temperature range used in the calculation and the sensitivity of the equipment to measure change in temperature at faster cooling rates. Table 5. Volume fractions of αp 940°C 910°C 880°C Oil Quench 4% 12% 25% Air Cool 7% 20% 29% Vermiculite Cool 8% 22% 37% With decreasing cooling rate, the αp volume fraction increased as grains coarsened and alpha stabilising elements had more time to partition to αp grains, seen in Figure 6. 4 MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032112019 The 14th World Conference on Titanium Figure 6. Effect of cooling rate on primary alpha volume fraction No strong relationships were observed between the volume fraction of αp and tensile strength for any of the cooling mediums, seen in Figure 7. This suggests that the base chemistry and the morphology of the higher volume fraction transformation product provides the primary impact on tensile properties rather than αp content in this alloy as illustrated by differences b li di Figure 6. Effect of cooling rate on primary alpha volume fraction erved between the volume fraction of αp and tensile strength for any of the cooling mediums, seen in Figure 7. This suggests that the base chemistry and lume fraction transformation product provides the primary impact on tensile properties rather than αp content in this alloy as illustrated by differences No strong relationships were observed between the volume fraction of αp and tensile strength for any of the cooling mediums, seen in Figure 7. This suggests that the base chemistry and the morphology of the higher volume fraction transformation product provides the primary impact on tensile properties rather than αp content in this alloy as illustrated by differences between cooling medium. Figure 7. MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium Effect of primary alpha volume fraction on proof strength of Ti575 Figure 7. Effect of primary alpha volume fraction on proof strength of Ti575 Figure 7. Effect of primary alpha volume fraction on proof strength of Ti575 Figure 7. Effect of primary alpha volume fraction on proof strength of Ti575 HCF HCF Table 5. HCF results in the solution heat treated and aged condition. 880°C VermC was omitted due to fracture location in the thread. Specimen Identity Achieved Frequency (Hz) Cycles Completed Fracture Location 880°C OQ 106 4626960 A - mid 1/3 880°C AC 106 8330725 A - mid 1/3 910°C OQ 106 6696087 A - mid 1/3 910°C AC 106 7631276 B - outer 1/3 (top) 910°C VermC 105 5553646 B - outer 1/3 (top) 940°C OQ 106 6545598 B - outer 1/3 (bottom) 940°C AC 107 4019967 B - outer 1/3 (top) 940°C VermC 106 1593800 B - outer 1/3 (bottom) Table 5. HCF results in the solution heat treated and aged condition. 880°C VermC was omitted due to fracture location in the thread. 5 MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032112019 Figure 8. Peak Stress vs Cycles to Failure for Ti575 tested at room temperature, R=0, ~100Hz Sinusoidal Waveform The sample solution heat treated at 880°C and vermiculite cooled fracture in the thread during HCF testing and this data point was excluded. Figure 9 indicates that as the αp volume fraction increased the number of cycles to failure increased for both the air and vermiculite cooled tests. This is explained in more detail in the EBSD section. However, for the oil quenched sample this was not the case showing a drop in the number of cycles to failure at larger volume fractions of αp. This could be related to the change in morphology associated with oil quenching and the high dislocation density of martensite providing more strength than αp grains therefore, the lower volume fraction αp in this microstructural condition provided better HCF results. Wu et al [6] found that in bi-modal Ti-6Al-4V microstructures αp volume fractions between 30-50% provided the best fatigue performance which agreed with the trend seen for air and vermiculite cooled tests. Repeat testing to provide additional data points are necessary to confirm the trends in the HCF data as variability between tests are common. Figure 8. MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium Peak Stress vs Cycles to Failure for Ti575 tested at room temperature, R=0, ~100Hz Sinusoidal Waveform Figure 8. Peak Stress vs Cycles to Failure for Ti575 tested at room temperature, R=0, ~100Hz Sinusoidal Waveform The sample solution heat treated at 880°C and vermiculite cooled fracture in the thread during HCF testing and this data point was excluded. Figure 9 indicates that as the αp volume fraction increased the number of cycles to failure increased for both the air and vermiculite cooled tests. This is explained in more detail in the EBSD section. However, for the oil quenched sample this was not the case showing a drop in the number of cycles to failure at larger volume fractions of αp. This could be related to the change in morphology associated with oil quenching and the high dislocation density of martensite providing more strength than αp grains therefore, the lower volume fraction αp in this microstructural condition provided better HCF results. Wu et al [6] found that in bi-modal Ti-6Al-4V microstructures αp volume fractions between 30-50% provided the best fatigue performance which agreed with the trend seen for air and vermiculite cooled tests. Repeat testing to provide additional data points are necessary to confirm the trends in the HCF data as variability between tests are common. Figure 9. αp Volume Fraction vs Cycles to Failure for Ti575 cooled using different mediums αp Volume Fraction vs Cycles to Failure for Ti575 cooled using different mediums EBSD EBSD Previous work carried out by Gey et al [4] determined that after transformation there are 5 misorientation types possible for variants within the same parent beta grain. By comparing misorientation of neighbouring pixels with theoretical misorientations, prior beta grain boundaries and αs colonies can be determined. In Figure 10, white lines denote the misorientations of adjacent pixels which match theoretical values (indicating αs colony sizes) and black lines denote any other misorientation provided their misorientation angles are higher than 3° (indicating grain boundaries). At higher volume fractions of αp, smaller prior beta grains were observed, due to an increase in grain boundary pinning. This reduced grain size did not have a strong impact on tensile strength but appeared to reduce the number of cycles to failure during HCF. At higher volume fractions of αp, smaller prior beta grains were observed, due to an increase in grain boundary pinning. This reduced grain size did not strength but appeared to reduce the number of cycles to failure during HCF. of αp, smaller prior beta grains were observed, due to an increase in grain boundary pinning. This reduced grain size did not have a strong impact on tensil ce the number of cycles to failure during HCF. Faster cooling rates resulted in smaller αs colony sizes which increased the tensile strength due to an increase in dislocation density. Slower cooling rates led to coarser lathes with increased local texture which might have contributed to reduction of cycles to failure observed for air and vermiculite cooled samples during HCF testing. Larger prior beta grain size (seen at lower αp volume fractions) led to larger αs colonies, which in turn caused larger areas of local texture. This increased texture again might have contributed to the decrease in number of cycles to failure observed for air and vermiculite cooled samples during HCF testing. 6 MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032112019 7 5. Further Work Repeat HCF testing for sample excluded for fracturing in the thread, duplicate testing to confirm trends in HCF data and to determine whether high αp volume fraction oil quench test was an anomaly. Separate αp and αs pole figures on EBSD maps to determine whether αs which grow from αp grains share similar orientation to help identify a link between αs variants aligning their c-axis with αp. 7 MATEC Web of Conferences 321, 12019 (2020) The 14th World Conference on Titanium MATEC Web of Conferences 321, 12019 (2020) https://doi.org/10.1051/matecconf/202032112019 The 14th World Conference on Titanium Figure 10. High resolution EBSD maps and prior beta grain boundaries for Ti575 SHT at a) 880°C and air cooled b) 910°C and air cooled c) 940°C and air cooled d) 940°C and oil quenched e) 940°C and vermiculite cooled Figure 10. High resolution EBSD maps and prior beta grain boundaries for Ti575 SHT at a) 880°C and air cooled b) 910°C and air cooled c) 940°C and air cooled d) 940°C and oil quenched e) 940°C and vermiculite cooled 4. Conclusions In this work, the cooling rates required to form martensitic, basketweave and aligned αs morphologies for Ti575 were established along with the temperature ranges in which key microstructural features begin to form when air cooling from the β phase. The base chemistry and the morphology of the higher volume fraction transformation product appeared to provide the primary impact on tensile properties rather than αp content in this alloy. With increasing αp volume fraction the number of cycles to failure during HCF for air and vermiculite cooled samples increased. However, a drop in the number of cycles to failure during HCF for the oil quenched test at high volume fraction of αp was observed, this is believed to be related to the change in morphology of the transformation product. hanges in morphology with smaller αs colonies which increased the tensile strength. Faster cooling rates lead to changes in morphology with smaller αs colonies which increased the tensile strength. EBSD showed that lower volume fraction of αp and slower cooling rates led to larger coarser αs colonies which in turn lead to larger textured regions which might be contributing to a reduction in the number of cycles to failure during HCF testing. 6. References [1] Rugg et al, “Effective structural unit size in titanium alloys,” Journal of Strain analysis, Vol. 42, 2007. [1] Rugg et al, “Effective structural unit size in titanium alloys,” Journal of Strain analysis, Vol. 42, 2007. [2] Thomas et al. “TIMETAL 575: A Novel High Strength Forgeable α/β Titanium Alloy,” Conference proceedings of the 13th World titanium Conference, 2016. [3] Sieniawski et al, “Microstructure and Mechanical Properties of High Strength Two-Phase Titanium Alloys,” Titanium Alloys - Advances in Properties Control, Chapter 4, 2013 [2] Thomas et al. “TIMETAL 575: A Novel High Strength Forgeable α/β Titanium Alloy,” Conference proceedings of the 13th World titanium Conference, 2016. [3] Sieniawski et al, “Microstructure and Mechanical Properties of High Strength Two-Phase Titanium Alloys,” Titanium Alloys - Advances in Properties Control, Chapter 4, 2013. [4] Gey et al, “Specific analysis of EBSD data to study the texture inheritance due to the β→α phase transformation,” Journal of materials science, Vol 38, Issue 6, 1289-1294, 2003. [5] L.Germain et al, “β→αs variant selection in sharp hcp textured regions of a bimodal IMI834 billet,” Textures of materials, Proceedings of the 14th International Conference of Textures of Materials, 2005. Thomas et al. “TIMETAL 575: A Novel High Strength Forgeable α/β Titanium Alloy,” Conference proceedings of the 13th World titanium Conference, 2016 [4] Gey et al, “Specific analysis of EBSD data to study the texture inheritance due to the β→α phase transformation,” Journal of materials science, Vol 38, Issue 6, 1289-1294, 2003. [5] L.Germain et al, “β→αs variant selection in sharp hcp textured regions of a bimodal IMI834 billet,” Textures of materials, Proceedings of the 14th International Conference of Textures of Materials, 2005. [5] L.Germain et al, “β→αs variant selection in sharp hcp textured regions of a bimodal IMI834 billet,” Textures of materials, Proceedings of the 14th International Conference of Textures of Materials, 2005. [6] Wu et al, “Effect of microstructure on the fatigue properties of Ti-6Al-4V titanium alloys,” Materials and Design, Vol 46, 668-674, 2013. 8 8
https://openalex.org/W1532027655	https://europepmc.org/articles/pmc2851500?pdf=render	English	null	Low-Pathogenicity Influenza Virus	Emerging infectious diseases	2,007	cc-by	2,942	Philip I. Marcus,* Theodore Girshick,† Louis van der Heide,* and Margaret J. Sekellick* Philip I. Marcus,* Theodore Girshick,† Louis van der Heide,* and Margaret J. Sekellick* Chicken interferon-α administered perorally in drink- ing water acts on the oropharyngeal mucosal system as an adjuvant that causes chickens to rapidly seroconvert after natural infection by low-pathogenicity Inﬂ uenza virus. These chickens, termed super sentinels, can serve as sensitive early detectors of clinically inapparent infections. Three-week-old speciﬁ c-pathogen-free (SPF) white leg- horns (Charles River Speciﬁ c Pathogen Free Avian Supplies [SPFAS], Inc., Storrs, CT, USA) were tagged and divided into 2 groups of 10 chickens each. Two birds in each group were overtly infected intravenously or intranasally with 106 infectious particles, measured as plaque-forming particles in primary chicken kidney cells (Charles River SPAFAS, Inc.). This strain of LPAI (H7N2) required a high inoculum to en- sure infection (data not shown), comparable to that reported for another LPAI (H7N2) strain evaluated in SPF chickens (9). The 8 remaining cage mates in each group served as sen- tinel birds naturally subject to infection by the respiratory tract, ingestion of fecal material, or both. One group of birds received plain drinking water; the other group received drink- ing water that contained 2,000 U/mL rChIFN-α. The water was provided ad libitum and changed daily. Water consump- tion was the same in both groups, as determined from the amount remaining after a known volume was provided each day (data not shown). With a half-life of 3–5 days in water at room temperature (6), this concentration of interferon (IFN) delivered an average dose of ≈3 × 105 U rChIFN-α/bird/day. Fourteen days post overt infection (dpi), the rChIFN-α-water was replaced with plain water for the remaining 14 days of the study. This dose of rChIFN-α was sufﬁ cient to amelio- rate Newcastle disease (6). E arly detection of low-pathogenicity type A inﬂ uenza virus (LPAI) circulating among chickens is impor- tant for 3 reasons: 1) these are the most prevalent strains in nature and can cause substantial losses for commercial poultry producers (1), 2) these strains can contribute ge- netic material to high-pathogenicity type A inﬂ uenza virus (HPAI) (2), and 3) the H5 and H7 LPAI strains can mutate to HPAI with catastrophic effects in birds and with the po- tential for transmission to humans with lethal consequences (3). Kuiken et al. Philip I. Marcus,* Theodore Girshick,† Louis van der Heide,* and Margaret J. Sekellick* reported that an HPAI (H7N7) isolate was observed in February 2003 in the Netherlands, which most likely originated in free-living ducks and had evolved into a highly pathogenic variant after introduction into poul- try farms (4). Although subsequent serologic screening of poultry showed that the H7 inﬂ uenza virus had been affect- ing the Dutch poultry industry several months before the major epidemic, its presence had not been recognized (4). Our study addresses this problem by using a novel method that causes chickens to seroconvert under conditions in which LPAI would otherwise go undetected. This report shows that recombinant chicken interferon-α (rChIFN-α) (5) administered perorally in drinking water (6) acts as an adjuvant to produce a super-sentinel chicken that is a sensi- tive and early detector of clinically inapparent LPAI. E Following overt infection of 2 birds per cage, and the natural cross-infection of the 8 cage mates, serum samples were taken from each of the 10 birds at the intervals in- dicated in Figure 1. This ﬁ gure shows data from 2 inde- pendent studies that used agar gel precipitin (AGP) tests to detect antibody against avian inﬂ uenza virus nucleoprotein and M1 antigens. This qualitative test demonstrated that of the 16 naturally infected chickens given plain water, none seroconverted over the 28-day period they were exposed to the 2 infected cage mates. In marked contrast, of the 16 naturally infected chickens given water containing IFN, 14 DISPATCHES DISPATCHES University of Connecticut, Storrs, Connecticut, USA; and †Charles River Speciﬁ c Pathogen Free Avian Supplies, Storrs, Connecticut, USA Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 10, October 2007 Super-Sentinel Chickens and Detection of Low-Pathogenicity Inﬂ uenza Virus to a diagnostic laboratory to conﬁ rm the diagnosis of LPAI (H7N2) infection at National Veterinary Services Labora- tory (NVSL) (N. Adriatico, pers. comm.). One such iso- late, A/CK/CT/72/2003(H7N2), was obtained from the US Department of Agriculture, NVSL, Ames, Iowa, and used throughout this study to determine whether the peroral ad- ministration of rChIFN-α under conditions found to ame- liorate Newcastle disease (6), infectious bronchitis (7), and infectious bursal disease (8), would similarly affect avian inﬂ uenza. We reasoned that if the spread of LPAI could be slowed or prevented, the probability of its mutating to HPAI would be proportionately reduced, thereby lowering the chances of transmission to humans. In the course of this study, we observed a strong adjuvant effect of rChIFN-α ad- ministered in drinking water under conditions of virus trans- mission that mimic natural infection in chickens. This led to the concept of the super-sentinel chicken described here. to a diagnostic laboratory to conﬁ rm the diagnosis of LPAI (H7N2) infection at National Veterinary Services Labora- tory (NVSL) (N. Adriatico, pers. comm.). One such iso- late, A/CK/CT/72/2003(H7N2), was obtained from the US Department of Agriculture, NVSL, Ames, Iowa, and used throughout this study to determine whether the peroral ad- ministration of rChIFN-α under conditions found to ame- liorate Newcastle disease (6), infectious bronchitis (7), and infectious bursal disease (8), would similarly affect avian inﬂ uenza. We reasoned that if the spread of LPAI could be slowed or prevented, the probability of its mutating to HPAI would be proportionately reduced, thereby lowering the chances of transmission to humans. In the course of this study, we observed a strong adjuvant effect of rChIFN-α ad- ministered in drinking water under conditions of virus trans- mission that mimic natural infection in chickens. This led to the concept of the super-sentinel chicken described here. Conclusions Although the role of IFN as an adjuvant when deliv- ered perorally has been established in mammals (12), our data demonstrate for the ﬁ rst time, to our knowledge, that avian IFN administered in drinking water to naturally in- fected chickens lowers the threshold of antigen required to stimulate the adaptive immune response to an LPAI iso- late. As a consequence, the action of perorally administered rChIFN-α in effect creates super-sentinel chickens that se- roconvert in response to levels of antigen that would oth- erwise go undetected. Super-sentinel chickens would thus provide a novel means of detecting otherwise inapparent infections of LPAI, thereby buying time for its control or eradication. were seropositive by 14 dpi and remained so during the 28-day test period. Figure 2 shows the number of seroconverted birds in a third study as quantiﬁ ed by hemagglutination inhibition (HI) titer (HI U/mL) of serum samples taken at the time intervals indicated as dpi. None of the 8 naturally infected birds given plain water seroconverted during the 28 days of the trial. In contrast, the 8 naturally infected chickens raised on IFN-water all seroconverted by 10 dpi (8/8), as did the overtly infected birds. Similar results were observed in 2 other trials. In all, 4 independent comparable trials were conducted, representing 2 AGP and 2 HI tests (Table 1). The marked contrast in the fraction of naturally infected birds that seroconverted on plain water and IFN-water is evident. Table 1. Seroconversion in influenza A virus–infected 3-week-old chickens given water or water + interferon No. chickens Infection type† Water Water + interferon‡ Total Overtly infected 4/8§ 8/8 16 Sentinel 2/32 31/31 63 Combined 6/40 39/39 79 Represents 4 independent trials. †Overtly infected birds were mixed with uninfected sentinel cage mates, the latter to become infected naturally. ‡Recombinant chicken interferon-α at 2,000 U/mL (5). §No. positive birds/total no. receiving treatment, scored by agar gel precipitin or hemagglutination inhibition tests. Table 1. Seroconversion in influenza A virus–infected 3-week-old chickens given water or water + interferon Although the sensitivity of LPAI to the action of IFN is well documented (10,11), rChIFN-α in the drinking water may have been exacerbating the infection, thereby leading to high levels of virus and antigen and high levels of sero- conversion. The Study In 2003, the ﬁ rst clue to an aberrant condition in a com- mercial ﬂ ock of laying hens in Connecticut was signaled by a drop in feed consumption and then in egg production. It took 6–7 weeks from the time tracheal samples were sent Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 10, October 2007 1608 Super-Sentinel Chickens and Inﬂ uenza Virus Figure 2. Seroconversion in sentinel speciﬁ c-pathogen-free white leghorns after natural infection with inﬂ uenza A/CK/CT/72/03 (H7N2) from overtly infected birds as quantiﬁ ed by hemagglutination inhibition (HI) tests for hemagglutinin (HA) antigen. The titer in HI U/mL is plotted as a function of days post overt infection of 2 birds in each group. The key is similar to that of Figure 1, except the assay is for HI. A, water only; B, water plus recombinant chicken interferon-α at 2,000 U/mL. Results of 1 trial are shown; 2 other trials gave similar results. Figure 2. Seroconversion in sentinel speciﬁ c-pathogen-free white leghorns after natural infection with inﬂ uenza A/CK/CT/72/03 (H7N2) from overtly infected birds as quantiﬁ ed by hemagglutination inhibition (HI) tests for hemagglutinin (HA) antigen. The titer in HI U/mL is plotted as a function of days post overt infection of 2 birds in each group. The key is similar to that of Figure 1, except the assay is for HI. A, water only; B, water plus recombinant chicken interferon-α at 2,000 U/mL. Results of 1 trial are shown; 2 other trials gave similar results. Figure 2. Seroconversion in sentinel speciﬁ c-pathogen-free white leghorns after natural infection with inﬂ uenza A/CK/CT/72/03 (H7N2) from overtly infected birds as quantiﬁ ed by hemagglutination inhibition (HI) tests for hemagglutinin (HA) antigen. The titer in HI U/mL is plotted as a function of days post overt infection of 2 birds in each group. The key is similar to that of Figure 1, except the assay is for HI. A, water only; B, water plus recombinant chicken interferon-α at 2,000 U/mL. Results of 1 trial are shown; 2 other trials gave similar results. Figure 1. Seroconversion in speciﬁ c-pathogen-free white leghorns after infection with inﬂ uenza A/CK/CT/72/2003 (H7N2) as measured by agar gel precipitin (AGP) tests for avian inﬂ uenza virus nucleo- protein and M1 antigens. Each box represents 1 chicken; (–), water; (+), water plus recombinant chicken interferon-α at 2,000 U/mL. IFN, interferon; IN, intranasal; IV, intravenous. The Study A and B are independent trials. Serum samples were obtained at the times indicated on days post infection for overtly infected birds. from testing individual chickens, the amount of infectious particle equivalents were not signiﬁ cantly different in birds given plain water or IFN-water. Thus, that more avian in- ﬂ uenza virus antigen was produced in chickens that were given IFN-water is an unlikely explanation. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 10, October 2007 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 10, October 2007 1609 References 1. Cardona C. Low-pathogenicity avian inﬂ uenza virus outbreaks in commercial poultry in California. In: The threat of pandemic inﬂ u- enza: are we ready? Washington: National Academies Press; 2005. p. 243–53. interferon and infected naturally Day postinfection Water (IPE/mL) Water + interferon† (IPE/mL) 2 1,112 ± 1,353‡ 760 ± 632 4 1,234 ± 764 463 ± 484 10 1,325 ± 398 2,113 ± 1,834 Each cage contained 2 overtly infected birds and 8 cage mates as sentinels. Only sentinel birds are reported. Chickens were 3 weeks old at the start. †Recombinant chicken interferon-α in water at 2,000 U/mL (5). ‡Mean ± SD, n = 8. Quantitative real-time reverse transcriptase–PCR analysis with influenza A virus standard: IPE/mL. 2. Chin PS, Hoffman E, Webby R, Webster RG, Guan Y, Peiris M, et al. Molecular evolution of H6 inﬂ uenza viruses from poultry in south- eastern China: prevalence of H6N1 inﬂ uenza viruses possessing seven A/Hong Kong/156/97 (H5N1)–like genes in poultry. J Virol. 2002;76:507–16. Each cage contained 2 overtly infected birds and 8 cage mates as sentinels. Only sentinel birds are reported. Chickens were 3 weeks old at the start. 3. Lee C-W, Swayne DE, Linares JA, Senns DA, Suarez DI. H5N2 avi- an inﬂ uenza outbreak in Texas in 2004: the ﬁ rst highly pathogenic strain in the United States in 20 years? J Virol. 2005;79:11412–21. 3. Lee C-W, Swayne DE, Linares JA, Senns DA, Suarez DI. H5N2 avi- an inﬂ uenza outbreak in Texas in 2004: the ﬁ rst highly pathogenic strain in the United States in 20 years? J Virol. 2005;79:11412–21. †Recombinant chicken interferon-α in water at 2,000 U/mL (5). ‡Mean ± SD, n = 8. Quantitative real-time reverse transcriptase–PCR analysis with influenza A virus standard: IPE/mL. 4. Kuiken T, Leighton FA, Fouchier RAM, LeDuc JW, Peitis JSM, Schdel A, et al. Pathogen surveillance in animals. Science. 2005;309:1680–1. We envision the introduction into a large ﬂ ock of a number of small cages containing chickens in which IFN- water replaces plain water. These super-sentinel chickens will serve as sensitive early detectors of LPAI, like the pro- verbial canary used in mines to detect low levels of toxic gases. Because of the cross-reaction between chicken and turkey IFN-α (5,13), super-sentinel turkeys could likely be created in a similar manner. Super-sentinel birds could be replaced every month and possibly returned to production. 5. Sekellick MJ, Ferrandino AF, Hopkins DA, Marcus PI. References Chicken in- terferon gene: cloning, expression, and analysis. J Interferon Res. 1994;14:71–9. 6. Marcus PI, van der Heide L, Sekellick MJ. Interferon action on avi- an viruses. I. Oral administration of chicken interferon-α ameliorates Newcastle disease. J Interferon Cytokine Res. 1999;19:881–5. Newcastle disease. J Interferon Cytokine Res. 1999;19:88 7. Pei J, Sekellick MJ, Marcus PI, Choi I-S, Collisson EW. Chicken interferon type I inhibits infectious bronchitis virus (IBV) replica- tion and associated respiratory illness. J Interferon Cytokine Res. 2001;21:1071–7. All strains of chickens tested, including those in the People’s Republic of China, have proved to be sensitive to the action of rChIFN-α (14). Genetically engineered pro- duction of rChIFN-α (15), treatment with it optimized for dose and duration, and its long half-life in water may make it economically feasible to convert many birds in a ﬂ ock to super-sentinel status. It also may be prudent to set up super- sentinel birds in areas of high risk for avian inﬂ uenza virus outbreaks, such as live-bird markets. Surveillance of other families of birds might be possible with species-speciﬁ c IFN. Further studies are required to test these possibilities and the extent to which rChIFN-α functions as an adjuvant with other strains of avian inﬂ uenza virus and chickens. 8. Mo CW, Cao TC, Lim BL. The in vivo and in vitro effects of chicken interferon-alpha on infectious bursal disease virus and Newcastle disease infection. Avian Dis. 2001;45:389–99. 9. Lu H, Castro AE. Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian inﬂ u- enza virus in speciﬁ c-pathogen-free chickens. Avian Dis. 2004;48: 263–70. 10. Sekellick MJ, Carra SA, Bowman A, Marcus PI. Transient resistance of inﬂ uenza virus to interferon action attributed to random packag- ing and activity of NS genes. J Interferon Cytokine Res. 2000;20: 963–70. 11. Cauthen AN, Swayne DE, Sekellick MJ, Marcus PI, Suarez DL. Amelioration of inﬂ uenza pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene. J Virol. 2007;81:1838–47. 12. Beilharz MW, Fleischmann WR Jr, editors. Special topics issue: oral use of interferons and cytokines. J Interferon Cytokine Res. 1999;19:813–979. Conclusions This possibility was tested by using quantita- tive real-time reverse transcriptase–PCR to determine the amount of avian inﬂ uenza virus in tracheal samples at 2, 4, and 10 dpi. Table 2 shows that within the error expected ng Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 10, October 2007 1609 1609 DISPATCHES Table 2. Influenza A virus infectious particle equivalents (IPE) in tracheal swabs from sentinel chickens given water or water + interferon and infected naturally* Address for correspondence: Philip I. Marcus, Department of Molecular and Cell Biology, University of Connecticut, U-3125, 75 North Eagleville Rd, Storrs, CT 06269, USA; email: philip.marcus@uconn.edu Address for correspondence: Philip I. Marcus, Department of Molecular and Cell Biology, University of Connecticut, U-3125, 75 North Eagleville Rd, Storrs, CT 06269, USA; email: philip.marcus@uconn.edu Acknowledgments We thank David L. Suarez for the quantitative real-time reverse transcriptase–PCR assays. 13. Suresh M, Karaca K, Foster D, Sharma JM. Molecular and function- al characterization of turkey interferon. J Virol. 1995;69:8159–63. 14. Xia C, Liu J, Wu ZG, Lin CY, Wang M. The interferon-alpha genes from three chicken lines and its effects on H9N2 inﬂ uenza viruses. Anim Biotechnol. 2004;15:77–88. This research was supported by US Department of Agricul- ture grant 58-1940-0-007 through the Center of Excellence for Vaccine Research at the University of Connecticut. The study ben- eﬁ ted from the use of the Animal Cell Culture Facility of the Bio- technology/Bioservices Center at the University of Connecticut. 15. Schultz U, Rinderle C, Sekellick MJ, Marcus PI, Staeheli P. Recom- binant chicken interferon from Escherichia coli and transfected COS cells is biologically active. Eur J Biochem. 1995;229:73–6. Dr Marcus is professor of molecular and cell biology at the University of Connecticut, Storrs. He has a long-standing interest in the chicken interferon system, including its cloning, expression in the developing embryo, and the sensitivity of inﬂ uenza viruses to the antiviral action of interferon. 1610 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 10, October 2007
https://openalex.org/W2971755588	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0222089&type=printable	English	null	Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions	PloS one	2,019	cc-by	10,137	RESEARCH ARTICLE Editor: Maria Lina Tornesello, Istituto Nazionale Tumori IRCCS Fondazione Pascale, ITALY Editor: Maria Lina Tornesello, Istituto Nazionale Tumori IRCCS Fondazione Pascale, ITALY Received: February 4, 2019 Accepted: August 21, 2019 Published: September 5, 2019 Copyright: © 2019 Shukla 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. Funding: The study was supported by research grants from ICMR (sanction no.- 5/13/38/2014- NCD-III), and DST-SERB (EMR/2017/004018), and DBT extramural grant (6242-P34/RGCB/PMD/DBT/ ALCB/2015) to ACB & ICMR-Senior Research Fellowship to MJ (3/2/2/278/2014-NCD III). Intramural funding from Delhi University (DST – PURSE Phase II/RC/2016/944) and ICMR to ACB is OPEN ACCESS Citation: Shukla S, Jadli M, Thakur K, Shishodia G, Mahata S, Basir SF, et al. (2019) Level of phospho- STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions. PLoS ONE 14(9): e0222089. https://doi.org/10.1371/ journal.pone.0222089 Shirish Shukla1¤a, Mohit Jadli2, Kulbhushan ThakurID2, Gauri Shishodia1¤b, Sutapa Mahata1¤c, Seemi Farhat Basir3, Bhudev Chandra Das4, Alok Chandra BhartiID1,2* 1 Division of Molecular Oncology, National Institute of Cancer Prevention and Research Noida, Uttar Pradesh, India, 2 Molecular Oncology Laboratory, Department of Zoology, University of Delhi, Delhi, India, 3 Department of Biosciences, Jamia Millia Islamia, New Delhi, India, 4 Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR) Amity University, Noida, Uttar Pradesh, India a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤a Current address: Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America. ¤b Current address: Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America. ¤c Current address: Chittaranjan National Cancer Institute, Kolkata, West Bengal, India. * alokchandrab@yahoo.com Abstract Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determi- nants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it’s association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues har- boring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetect- able pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promo- tion/maintanence of tumorigenic phenotype. Editor: Maria Lina Tornesello, Istituto Nazionale Tumori IRCCS Fondazione Pascale, ITALY Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions Shirish Shukla1¤a, Mohit Jadli2, Kulbhushan ThakurID2, Gauri Shishodia1¤b, Sutapa Mahata1¤c, Seemi Farhat Basir3, Bhudev Chandra Das4, Alok Chandra BhartiID1,2* Competing interests: The authors declare that there are no conflicts of interests. Abbreviations: HPV, human papillomavirus; HSIL, high grade squamous intraepithelial lesions; LSIL, low grade squamous intraepithelial lesions; SCC, squamous cell carcinoma; pSTAT3, phospho- tyrosine705-signal transducer and activator of transcription; URR, upstream regulatory region. Expression of HPV genome depends primarily on host transcription factors that work on specific enhancer regions present in HPV Upstream Regulatory or Long Control Region (URR/LCR) [20]. A set of transcription factors like STAT3, AP-1, NF-κB, SP1, NF-1, c/EBP, Oct-1, KRF-1, YY1, and GRE have been proposed to play a regulatory role in HPV infection due to the presence of their cognate cis-elements in the URR [20–22]. These factors are respon- sible for the cell-type-specific viral gene expression and contribute to the tissue tropism of HPVs [20,23]. Apart from critically influencing the viral oncogenes expression, host transcrip- tion factors directly influence other important determinants of HPV infection such as viral replication [24,25] and may be involved in viral integration in host cell genome via induction of genomic instability [26]. Characterization of host cell transcription factors has revealed a disease stage and grade-specific expression and activity of some of the key transcription factors like AP-1, NF-κB, and STAT3 in cervical cancer carcinogenesis [27–29]. However, any influ- ence/association of these transcription factors on the maintenance of the HPV genome in an infected cell remains elusive. Therefore, improved understanding of molecular dynamics of specific host transcription factors expression and their correlation with the HPV genome phys- ical state and copy number in cervical carcinogenesis is required to develop effective infection- specific biomarkers and the therapeutic targets. Assessment of promoter activity in HPV16 [30] and ChIP-sequencing data of HPV18 [22] revealed presence of binding sites for STAT3 in URR, which plays a pivotal role in epithelial carcinogenesis [31]. STAT3 expression and activation is known to increase with disease sever- ity [29] and has been shown to contribute functionally by regulating expression of viral onco- gene E6 [32]. However, the functional relevance of STAT3 concerning the viral load and physical state of the HPV genome in the host cell remained unexplored. STAT3 is an inducible transcription factor that works as an important link between inflammation and carcinogenesis [33]. Its activity and nuclear translocation are controlled by specific phosphorylation at tyro- sine 705 [pSTAT3(Y705)] that results in its dimerization, nuclear translocation, and DNA binding [33]. Evidence from our group [34] and others [35], indicate the presence of active STAT3 in cervical cancer stem cells. Introduction Progression to cervical cancer is a multi-step process etiologically-linked with persistent infec- tion of high-risk human papillomaviruses (HPVs). Though HPV infection is a necessary 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis prerequisite, but it is not sufficient for the initiation of cervical cancer [1]. The physical state of the viral genome (integrated vs. episomal) and viral copy number in infected tissues have been evaluated as the candidate surrogate markers for the early detection of high grade and poten- tially progressive lesions, which showed a predictive potential of these biomarkers [2–4]. Viral integration often leads to the disruption of its E2 gene [5]. Loss of functional E2 gene abolishes the transcription-repressive effect of E2 on the expression of viral oncogenes E6 and E7 [6]. Absence of E2 due to insertional inactivation or by epigenetic silencing [7] and expression of E6 and E7 collectively drive the process of carcinogenesis [8,9]. On the contrary, integration of the HPV genome in low-grade lesions and occasionally in normal cervical tissues has also been reported [10,11], whereas, not all invasive cancers carry the integrated HPV genome [12– 14]. Clinical implication and the reasons behind such discrepant observations, particularly the confounding factors responsible for the phenomenon, are not clear as yet. Despite these varia- tions, high-risk HPV viral load and physical state are proposed as potentially useful markers that could predict progressive high-grade cervical lesions [15–17]. These markers, however, display differential type-dependent risks [18]. Nevertheless, in the case of HPV16 infection, these parameters (viral load and integration) consistently showed increased risk [4,19]. There- fore, a better understanding of factors that control these viral attributes will improve the per- formance of viral infection-specific biomarkers in predicting cervical disease progression, and subsequently the therapeutic outcome. also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that there are no conflicts of interests. Clinical specimens and reagents Out of 252 specimens, 130 HPV16 positive cervical tissues consisting of 60 pre-cancers with abnormal cytopathological diagnosis [LSIL (30) or HSIL (30)], and 70 cancer tissues qualified for analysis of viral load and the physical state of HPV16 by PCR-based method and pSTAT3 expression using western blotting (Table 1). A portion of each biopsy collected in cold 1X phosphate buffer saline (PBS) was immediately processed for molecular research work, and the other half was sent for routine histopathological diagnosis in formalin solution. All reagents used in the study were of analytical or molecular biology grade and procured from Sigma Aldrich (USA) unless specified. Custom-synthesized, HPLC-purified primers were pro- cured from either M/s Microsynth (Germany) or M/s Eurogentec (Belgium). Primers used in the study are listed in Table 2 [37–39]. Ethics statement A total of 252 fresh cervical biopsies were collected prospectively comprising of cervical tissues before any chemo-/radio-therapy from the Cancer Clinic, Gynae Out Patient Department of Lok Nayak Hospital, New Delhi, India. The age of participants ranged between 23 to 80 years. Written informed consent was obtained from all the participants included in the study and was carried out by the Principles of the Helsinki Declaration, and clinico-epidemiological details were taken from their clinical records. The study was approved by the Institutional Eth- ics Committee of the Institute of Cytology and Preventive Oncology (now renamed as National Institute of Cancer Prevention and Research), Noida, Utter Pradesh, India. Competing interests: The authors declare that there are no conflicts of interests. A recent study carried out on keratinocytes harboring HPV18 episomes demonstrated an essential role of active STAT3 in the maintenance of viral genome [36]. However, a clinical correlation of this experimental observation was lacking. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 2 / 16 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis In the present study, we investigated the existence of a correlation between active STAT3 and physical state as well as the copy number of viral genome in HPV16 positive tumor tissues from cervical precancer and cancer lesions. Level of active STAT3 (pSTAT3) was measured by immunoblotting of total proteins isolated from different cytopathological grades of tumor tis- sues. In parallel, DNA isolated from the respective tissues was subjected to analysis of HPV16 viral load which was measured by the copy number analysis, and the physical state was exam- ined by measuring the ratio of amplification of E2 versus E6 gene regions in HPV16 genome. To maintain homogeneity of the analysis, and to avoid confounding variables, cervical precan- cer and cancer tissues having non-HPV16 or multiple infections were excluded from the study. pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis Table 1. Clinicopathological distribution of subjects enrolled in the study along with PCR-based analysis of HPV infection in their tumor tissues. Tissue type Diagnosis Number of samples Total HPV+ (HPV L1) (n/%age) HPV 16 (n/%age) HPV18 (n/%age) Others HPV (n/%age) Multiple infections Mean age (Years; ± SD) No. of HPV16 positive cases qualified Normal 32 2 (6%) 2 (6%) - - - 40.5 ± 8.2 - Pre-cancer 120 65 (54%) 60 (50%) 3 (2.5%) 2 (1.5%) - 37.4 ± 6.9 60 LSIL 70 33 (47%) 30 (43%) 2 (3%) 1 (1.5%) - 30 HSIL 50 32 (64%) 30 (60%) 1 (2%) 1 (2%) - 30 Cancer 100 96 (96%) 89 (89%) 7 (7%) 3 (3%) 4(4%) 51 ± 11.8 70 Histopathological grading WDSCC 55 52 (95%) 46 (84%) 5 (9%) 2 (4%) 2 (4%) 35 MDSCC 35 34 (97%) 33 (94%) 2 (9%) 1 (3%) 2 (6%) 25 PDSCC 10 10 (100%) 10 (100%) - - - 10 Includes 3 cases of multiple infections of HPV16 with HPV18. HPV- human papillomavirus, SCC- squamous cell carcinoma, LSIL- low grade squamous intraepithelial lesions, HSIL-high grade squamous intraepithelial lesions, WDSCC-well differentiated SCC, MDSCC-moderately differentiated SCC, PDSCC-poorly differentiated SCC. gical distribution of subjects enrolled in the study along with PCR-based analysis of HPV infection in their tumor tissue Table 1. Clinicopathological distribution of subjects enrolled in the study along with PCR-based analysis of HPV infect Includes 3 cases of multiple infections of HPV16 with HPV18. HPV- human papillomavirus, SCC- squamous cell carcinoma, LSIL- low grade squamous intraepithelial lesions, HSIL-high grade squamous intraepithelial lesions, WDSCC-well differentiated SCC, MDSCC-moderately differentiated SCC, PDSCC-poorly differentiated SCC. DNA extraction and diagnosis of HPV infection High molecular weight genomic DNA was isolated from precancerous and cancerous cervical biopsies by the standard phenol-chloroform and proteinase K digestion procedure. PCR amplification was performed following the procedure described earlier [29]. The initial HPV diagnosis was performed by using a pair of L1 consensus degenerate primers (MY09 and MY11) based PCR method described earlier. HPV16 typing was done by type-specific primers (Table 2). Subsequently, HPV16 positive samples were subjected to comprehensive HPV gen- otyping by PGMY-Reverse Line Blot, which can detect about 32 HPV types including all high- risk and low-risk types [40] and samples with only monotypic HPV infection were included in the study. 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 https://doi.org/10.1371/journal.pone.0222089.t002 HPV16 viral load determination by real-time quantitative PCR (qRT-PCR) Quantification of HPV16 viral copy number and measurement of input cellular DNA copies was performed as described earlier [2] with an iQ-Cycler system (Biorad, Hercules, CA, USA) using a recommended iQ SYBR green PCR supermix according to the manufacturer’s instruc- tion. HPV16 URR primers were used for HPV copy number calculation, and p53 exon5 prim- ers were used for input DNA copy number calculation. Since the amplicon size of p53 exon5 primer set was much closer to the HPV16 URR PCR product and had similar efficiency of amplification; we used this primer set to control host DNA input. Pre-calibrated WHO’s Table 2. Oligonucleotide primers used for HPV diagnosis, HPV16 typing, viral load quantitation (HPV16 URR) and E2: E6 PCR in the present study. Primer Nucleotide Position in HPV Amplicon size (bp) Primers Type Primer sequences Final Conc. (μM) References MY 09 and L1 Consensus 450 Forward 5’CGT CCM ARR GGA WAC TGATC-3’ 20 pmoles [39] MY 11 Reverse 5’-GCM CAG GGW CAT AAY AAT GC-3’ (M = A+C, W = A+T, Y = C+T, R = A+G) HPV16URR 7763–7781 217 Forward 5’-AAG GCC AAC TAA ATG TCA C-3’ 20 pmoles [37] 57–75 Reverse 5’-CTG CTT TTA TAC AA CCG G-3’ p53 Exon 5 463–482 184 Forward 5’-TAC TCC CCT GCC CTC AAC AA-3’ 20 pmoles [37] 534–562 Reverse 5’-CAT CGC TAT CTG AGC AGC GC-3’ HPV16 E2 2734~2753 1139 Forward 5'-AGG ACG AGG ACA AGG AAA A-3' 20 pmoles [37] 3853–3872 Reverse 5'-GGA TGC AGT ATC AAG ATT TG-3' HPV 16 E6 83–102 477 Forward 5’-GAA ACC GGT TAG TAT AAA AGC AGA C-3’ 20 pmoles [38] 540–559 Reverse 5’- AGC TGG GTT TCT CTA CGT GTT CT-3’ β-Globin 268 Forward 5’-GAA GAG CCA AGG ACA GGT AC-3’ 10 pmoles [37] Reverse 5’-CAA CTT CAT CCA CGT TAC ACC-3’ rs used for HPV diagnosis, HPV16 typing, viral load quantitation (HPV16 URR) and E2: E6 PCR in the present study. gnosis, HPV16 typing, viral load quantitation (HPV16 URR) and E2: E6 PCR in the present study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 4 / 16 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis HPV16 International Standard DNA (06/202) procured from National Institute of Biological Standards and Control (NIBSC), UK was used as a reference. HPV16 viral load determination by real-time quantitative PCR (qRT-PCR) Crude viral load or copy number of HPV16 genome in the clinical sample was calculated by the interpolation of standard curves of the dilution series generated by the Sequence Detection Software (iCycler iQ software version 3.0) of iCycler iQ real-time PCR detection system (Biorad, Hercules, USA). On the other hand, samples with viral loads higher than 50,000 copies/reaction were USA). On the other hand, samples with viral loads higher than 50,000 copies/reaction were diluted in water to bring it down to the range of the standard curve. The viral load values were normalized to input host diploid genomic DNA using p53 exon5 amplification calibrated with C33a genomic DNA standard (NIBSC) as indicated below: Normalized HPV16 viral load/unit host cell genome = HPV16 URR copy number/ num- ber of diploid host genomes. The normalized HPV16 viral copy numbers are expressed as the number of viral copies/unit host cell genome. HPV16 viral load determination by real-time quantitative PCR (qRT-PCR) Standard curves used to quantify HPV16 copy number were made with ten fold serial dilutions of the WHO HPV16 interna- tional standard containing 50,000, 5000, 500, 50 and 5, HPV16 DNA copies diluted in the background of C33a genomic DNA. Briefly, the reaction was performed in a final volume of 25μl containing 1X SYBR green super-mix with 0.25μM of HPV16 URR forward and reverse primers and 50ng of genomic DNA of test samples. The URR primers were selected for viral load quantitation as these are retained in both episomal and integrated forms of the HPV16 viral genome. The PCR amplification was performed as follows: 1 cycle of 96˚C for 3min, 40 cycles at 94˚C for 30sec, 55˚C for 30sec and 72˚C for 30sec with realtime measurement per- formed during amplification step (72˚C) at each cycle. Each real-time amplification was fol- lowed by a melt curve analysis for confirmation of predicted amplicon. Crude viral load or copy number of HPV16 genome in the clinical sample was calculated by the interpolation of standard curves of the dilution series generated by the Sequence Detection Software (iCycler iQ software version 3.0) of iCycler iQ real-time PCR detection system (Biorad, Hercules, USA) O h h h d l i h i l l d hi h h 50 000 i / i HPV16 International Standard DNA (06/202) procured from National Institute of Biological Standards and Control (NIBSC), UK was used as a reference. Standard curves used to quantify HPV16 copy number were made with ten fold serial dilutions of the WHO HPV16 interna- tional standard containing 50,000, 5000, 500, 50 and 5, HPV16 DNA copies diluted in the background of C33a genomic DNA. Briefly, the reaction was performed in a final volume of 25μl containing 1X SYBR green super-mix with 0.25μM of HPV16 URR forward and reverse primers and 50ng of genomic DNA of test samples. The URR primers were selected for viral load quantitation as these are retained in both episomal and integrated forms of the HPV16 viral genome. The PCR amplification was performed as follows: 1 cycle of 96˚C for 3min, 40 cycles at 94˚C for 30sec, 55˚C for 30sec and 72˚C for 30sec with realtime measurement per- formed during amplification step (72˚C) at each cycle. Each real-time amplification was fol- lowed by a melt curve analysis for confirmation of predicted amplicon. Results Level of pSTAT3 (Y705) was analyzed by immunoblotting in a total of 130 HPV16 positive cases comprising 60 pre-cancer (LSIL– 30; HSIL– 30) and 70 cancer tissues and a correlation was examined with respective viral load and physical state of HPV16 genome from corre- sponding cervical lesions (Fig 1). pSTAT3 immunoblotting in proteins isolated from cervical tumor tissues Isolation of total cellular proteins from fresh biopsies and immunoblot analyses were per- formed using pSTAT3 (Y705) and pSTAT3 antibodies (BD Biosciences, USA) as described previously [29]. Membranes were re-probed for β-actin as an internal loading control. The quantitative densitometric analysis of the bands was performed using Alpha Ease FC version 4.1.0 (Alpha Innotech). The expression level of proteins was quantitated on an arbitrary scale with respect to the β-actin expression where Strong (+++)–>50%; Medium (++)– 10–50%; Weak (+)–<10% of β-actin expression; and Nil (–)–not-detectable. Determination of physical state of HPV16 genome in cervical tissues An E2: E6 ratio with a value of 0 represented completely integrated HPV16 genome and value of 1 or higher repre- sented predominantly episomal viral genome, whereas values > 0 and < 1 indicated a mixed form of HPV16 DNA. where IDV indicates integrated densitometric values of DNA band of HPV16 E2 amplicon (IDV) or HPV16 E6 (IDV) amplicon of the plasmid and sample DNA. An E2: E6 ratio with a value of 0 represented completely integrated HPV16 genome and value of 1 or higher repre- sented predominantly episomal viral genome, whereas values > 0 and < 1 indicated a mixed form of HPV16 DNA. Statistical analysis The data analysis was performed using the statistical software SPSS version 17 and SigmaPlot v14.0 (Systat Software, Inc.). To determine the mean and median values of continuous vari- ables and standard errors of means, descriptive statistics were used. As the distribution of viral load and E2: E6 ratio significantly departed from approximate normality, non-parametric tests were used to compare the distribution of study measurements across the study groups. Mann Whitney U test was used to compare the distribution of viral load and physical state of HPV16 along with the status of pSTAT3 expression between different disease groups. p values of <0.05 were considered statistically significant. Association between the level of pSTAT3 expression, viral load, and physical status of HPV16 among different categories of tissues from precancer and cancer lesions was examined by non-parametric Spearman’s rank-order corre- lation coefficient. Cervical disease groups were converted to categorical variables based on their increasing severity. pSTAT3 expression was evaluated on 4 point intensity scale as men- tioned above, whereas, viral load and physical state (E2: E6 ratio) were evaluated as continuous variables. These variables were examined for significance and strength of the relationship between study parameters in overall study samples and between test groups. To evaluate the association between pSTAT3, viral load, and physical state of HPV16 One Way Analysis of Variance (ANOVA) on ranks were performed using Kruskal-Wallis test, and multiple pairwise comparison was performed using the Tukey Test. Chi-square test was performed between two levels of STAT3 expression with respect to the three different physical states of the HPV16 genome where applicable. Determination of physical state of HPV16 genome in cervical tissues The physical state of HPV16 genome was determined by PCR as described previously [2]. To determine HPV16 physical state, primers for full-length HPV16 E2 validated by our laboratory previously [37], were utilized to analyze the presence of intact E2 ORF that is disrupted or deleted in the integrated virus. Amplification of HPV16 E6, which is invariably retained in the integrated virus, was used as the denominator of total HPV16 DNA irrespective of its physical state of the virus. Briefly, genomic DNA of HPV16-positive cases was used to assess the pres- ence or absence of the HPV16 E2 gene, concerning the HPV16 E6 gene. Genomic DNA of test samples (50ng) was PCR amplified for HPV16 E2 and E6 in a 25μl reaction mixture containing 10mM Tris-HCl (pH 8.4), 50mM KCl, 1.5mM MgCl2, 125μM of each dNTPs (dATP, dGTP, dCTP, dTTP), 5pmol of oligonucleotide primers for either full-length HPV16 E2 or HPV16 E6 and 0.5U AmpliTaqGold DNA polymerase (Applied Biosystems, USA). The amplification was performed with an initial denaturation at 95˚C for 4 min, polymerization for 35 cycles of dena- turation at 95˚C for 30sec, annealing at 55˚C for 30sec and extension at 72˚C for 1min, which was extended for 5min at the final cycle (Applied Biosystems). The densitometric ratio of E2 and E6 amplicons was measured on AlphaDigiDoc using Alpha Ease FC version 4.1.0 (Alpha Innotech Corporation, USA) to determine the physical state of HPV16 for each sample. Densi- tometric ratios of E2: E6 amplicons of all clinical samples were normalized to E2: E6 ratio of vector-free HPV16 plasmid (a kind gift from Prof. H. zur Hausen, DKFZ, Germany) which was used as a reference for a pure episomal form of HPV16 genome and helped to normalize the variations in PCR efficiencies. DNA from SiHa cells (Procured from American Type Cul- ture Collection, USA) was used as a control for fully integrated DNA. The E2: E6 ratio in clini- cal samples with reference to the plasmid control was calculated by the following formula: Normalized E2: E6 ratio of clinical samples = (IDV E2: IDV E6) samples /(IDV E2: IDV E6) plasmid 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis where IDV indicates integrated densitometric values of DNA band of HPV16 E2 amplicon (IDV) or HPV16 E6 (IDV) amplicon of the plasmid and sample DNA. Association between pSTAT3 expression and HPV16 viral load Cellular proteins (40μg/lane) derived from representative cervical tissues with indicated physical state of HPV16 genome (episomal- Epi; mixed–Mix; or integrated–Int) and viral load either lower (Low) or higher (High) than the median value of respective stage of disease were examined for pSTAT3 (Y705) and STAT3 expression by immunoblotting. Blots were stripped and re-probed with β-actin to equate the variations in cellular protein input. https://doi.org/10.1371/journal.pone.0222089.g001 host genome) and were the highest in cancer tissues (629 GE/unit host genome). Both LSIL and HSIL tissues, in general, had a weaker pSTAT3 expression along with lower viral load compared to cancer tissues which expressed stronger pSTAT3 and had consistently higher HPV16 viral load (p<0.001). Analysis of viral load and pSTAT3 level within different stages of disease demonstrated higher viral loads in samples that expressed moderate or strong pSTAT3 expression (Table 3). In LSIL, the moderate or strong expression of pSTAT3 was associated with the higher median copy number of HPV16 (47.5 GE/unit host genome) in comparison to the LSIL that had a weak or undetectable pSTAT3 expression (27.5 GE/unit host genome; p- value < 0.05). Likewise, in HSIL, the median copy number associated with moderate or strong pSTAT3 expression (207.5 GE/unit host genome) was 3-fold higher than a median viral load of cases with nil or weak pSTAT3 expression (73.5 GE/unit host genome; p-value < 0.01). In cancer tissues, the strong pSTAT3 expression was associated with higher median viral copy number (845 GE/Unit host genome) than cases with weak pSTAT3 expression (219 GE/Unit host genome; p-value < 0.001) (Table 3). The results demonstrate a progressive increase in viral copies with higher STAT3 activity that increased with the increasing severity of cervical lesions. Association between pSTAT3 expression and HPV16 viral load Distribution of samples concerning their viral load and level of pSTAT3 expression in LSIL, HSIL, and cancer (SCC) groups is presented in Fig 2. The median values for HPV16 viral load increased with disease severity from LSIL (33 GE/unit host genome) to HSIL (116 GE/unit 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis pSTAT3 (Y705) STAT3 ß Actin LSIL Epi HSIL Epi Mix Int CaCx Epi Mix Int High Low High High Low High Low High Low High HPV16 Physical State HPV16 Viral Load Stage of Cervical Disease Fig 1. Status of pSTAT3, along with the physical state and viral load of HPV16 in different cervical pre-cancer and cancer tissues. Representative photograph showing levels of active pSTAT3(Y705) and total STAT3 in HPV16 positive cervical lesions. Cellular proteins (40μg/lane) derived from representative cervical tissues with indicated physical state of HPV16 genome (episomal- Epi; mixed–Mix; or integrated–Int) and viral load either lower (Low) or higher (High) than the median value of respective stage of disease were examined for pSTAT3 (Y705) and STAT3 expression by immunoblotting. Blots were stripped and re-probed with β-actin to equate the variations in cellular protein input. pSTAT3 (Y705) STAT3 ß Actin LSIL Epi HSIL Epi Mix Int CaCx Epi Mix Int High Low High High Low High Low High Low High HPV16 Physical State HPV16 Viral Load Stage of Cervical Disease Fig 1. Status of pSTAT3, along with the physical state and viral load of HPV16 in different cervical pre-cancer and cancer tissues. Representative photograph showing levels of active pSTAT3(Y705) and total STAT3 in HPV16 positive cervical lesions. Cellular proteins (40μg/lane) derived from representative cervical tissues with indicated physical state of HPV16 genome (episomal- Epi; mixed–Mix; or integrated–Int) and viral load either lower (Low) or higher (High) than the median value of respective stage of disease were examined for pSTAT3 (Y705) and STAT3 expression by immunoblotting. Blots were stripped and re-probed with β-actin to equate the variations in cellular protein input. Fig 1. Status of pSTAT3, along with the physical state and viral load of HPV16 in different cervical pre-cancer and cancer tissues. Representative photograph showing levels of active pSTAT3(Y705) and total STAT3 in HPV16 positive cervical lesions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 Association between the expression of pSTAT3 and integrated HPV16 genome in pre-cancer and cancer lesions Next, we examined the association of the physical state of HPV16 genome classified as pre- dominantly episomal, integrated; or mixed form (concomitant presence of both episomal and integrated) which was identified using normalized HPV16 E2: E6 ratio, with levels of pSTAT3 in cervical pre-cancer and cancer lesions. Interestingly, pre-cancer tissues harboring episomal form demonstrated similar lower levels of pSTAT3 expression as in cancer tissues harboring episomal form, whereas the intensity of pSTAT3 was found to be higher in pre-cancer and cancer lesions harboring either mixed or integrated form of HPV16 genome (Fig 1). Distribu- tion of normalized E2: E6 ratio on the basis of pSTAT3 expression pattern, demonstrated that in most of the LSIL tissues where the HPV16 genome was present in the episomal form (median E2: E6 ratio—1.0), pSTAT3 expression was found to be either absent or weak. On the other hand, a significant number of HSIL and cancer tissues harboring either mixed or PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 7 / 16 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis LSIL (n=30) HSIL (n=30) SCC (n=70) Fig 2. Distribution of HPV16 viral load and pSTAT3 levels in cervical pre-cancer and cancer lesions. Each circle represents individual LSIL, HSIL, or cancer tissues (SCC) with respective viral load and pSTAT3(Y705) level as determined by real-time PCR and immunoblotting. Viral load values are normalized per cell genome equivalent as described in Methods. 1Arbitrary level of STAT3 expression in immunoblotting: Strong = (+++), Moderate = (+ +), Weak = (+), Nil/undetectable = (-). The vertical red line and horizontal blue line represent median values of STAT3 expression and viral load in tissues of each disease stage, respectively. GE, genome equivalents. Fig 2. Distribution of HPV16 viral load and pSTAT3 levels in cervical pre-cancer and cancer lesions. Each circle represents individual LSIL, HSIL, or cancer tissues (SCC) with respective viral load and pSTAT3(Y705) level as determined by real-time PCR and immunoblotting. Viral load values are normalized per cell genome equivalent as described in Methods. 1Arbitrary level of STAT3 expression in immunoblotting: Strong = (+++), Moderate = (+ +), Weak = (+), Nil/undetectable = (-). The vertical red line and horizontal blue line represent median values of STAT3 expression and viral load in tissues of each disease stage, respectively. GE, genome equivalents. Association between the expression of pSTAT3 and integrated HPV16 genome in pre-cancer and cancer lesions integrated form of HPV16 genome showed the elevated expression level of activated STAT3 (Median normalized E2: E6 ratio– 0; Median pSTAT3 expression–Strong) (Fig 3). Evaluation of the physical state of HPV16 and pSTAT3 expression in tissues from LSIL and HSIL cases, however, did not reveal any association between pSTAT3 expression with the mixed or inte- grated state as lesions harboring mixed or integrated HPV16 genome expressed variable amounts of pSTAT3 (Table 4). On the other hand, stratification of total cancer cases with respect to HPV16 physical state and level of pSTAT3 expression revealed a significant associa- tion (p values <0.05 and <0.001, respectively) between high pSTAT3 expression and inte- grated or mixed state of HPV16 genome. pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis Table 3. Distribution of HPV16 viral load in HPV16 positive cervical precancer and cancer lesions with respect to the level of pSTAT3 expression. Tissue Type Parameter HPV16 Viral Load (GE/Unit Host Genome) p valuea pSTAT3 Nil / Weak pSTAT3 Moderate / Strong LSIL (n = 30) Median 27.5 47.5 <0.05 Mean ± SE 28.0 ± 3.3 45.1 ± 3.7 N 24 6 HSIL (n = 30) Median 73.5 207.5 <0.05 Mean ± SE 119.4 ± 55.0 395.1 ± 117.1 N 18 12 SCC (n = 70) Median 219 845.0 <0.001 Mean ± SE 251.4 ± 41.7 1766.1 ± 260.6 N 13 57 LSIL—Tissues derived from Low grade Squamous Intraepithelial Lesions; HSIL—Tissues derived from High grade Squamous Intraepithelial Lesions; SCC—Tissues derived from Squamous Cell Carcinoma; SE–Standard Error ap-value between pSTAT3 (Nil/Weak) vs. pSTAT3 (Moderate/High) in each disease stage as determined by Mann-Whitney U test. Bold type refers to statistically significant p-values V16 viral load in HPV16 positive cervical precancer and cancer lesions with respect to the level of pSTAT3 expression. LSIL—Tissues derived from Low grade Squamous Intraepithelial Lesions; HSIL—Tissues derived from High grade Squamous Intraepithelial Lesions; SCC—Tissues derived from Squamous Cell Carcinoma; SE–Standard Error ap-value between pSTAT3 (Nil/Weak) vs. pSTAT3 (Moderate/High) in each disease stage as determined by Mann-Whitney U test. Bold type refers to statistically significant p-values pSTAT3 harbored higher copy numbers of HPV16 genome (Med—25 vs. 47.5 GE/unit host genome; p-value = 0.012) whereas HSIL with episomal HPV16 showed an increased pSTAT3 expression was associated with higher viral copies (Median—68 vs. 126.5 GE/unit host genome; p-value = 0.01). However, no difference was observed in HPV16 viral load with respect to pSTAT3 expression in LSIL and HSIL cases with integrated or mixed HPV16 genome. On the other hand, cancer tissues where the HPV16 genome existed as mixed or inte- grated physical form showed a strong association between increased pSTAT3 and elevated viral loads (p-value<0.01). p Since this being a multifaceted study comparing the level of pSTAT3 with viral load and physical state of HPV16 in different grades of cervical neoplastic disease representing cancer progression, correlation of these variables was statistically examined by non-parametric test. HPV16 viral load, its physical state, and level of active pSTAT3 are mutually related Further, analysis of pSTAT3 expression with respect to the differential viral load in different physical states of the HPV16 genome revealed lower levels of pSTAT3 expression in samples with lower viral load and episomal form as compared to cervical lesions with either higher viral load and/or with integrated or mixed form of HPV16 genomes (Fig 1). Cumulative data presented in Table 5 revealed that LSILs with HPV16 episomes and having moderate or strong 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 Assessment of association between lesion grade, viral load, E2: E6 ratio and pSTAT3 was per- formed using Spearman’s rank correlation coefficient which revealed a positive correlation between lesion grade, viral load and pSTAT3 with highly significant p-values (Table 6). On the contrary, a strong negative correlation existed between E2: E6 ratio and lesion grade, viral load, or pSTAT3 expression. A Kruskal-Wallis H test showed that there was a statistically sig- nificant difference in the median values among the groups which is greater than it would be expected by chance (H = 425.2 with 3 degrees of freedom; P<0.001). Similarly, pairwise multi- ple comparisons between lesion grade, viral load, E2: E6 ratio and pSTAT3 showed highly sig- nificant p-values except in the case of pSTAT3 vs. lesion grade (Table 7). https://doi.org/10.1371/journal.pone.0222089.t003 q aPearson Chi-square p<0.001between two levels of STAT3 expression concerning the three different physical status of HPV16. Bold type refers to statistically significant p values. Discussion HPV viral load and physical state of the viral genome are important determinants of HPV infection, which influence the tumorigenic transformation of normal cervical epithelium and progression of the disease [2,4,41,42]. Our earlier observations showed elevated STAT3 signal- ing in HPV-infected precancer and cancer lesions [43] and its functional contribution in cervi- cal carcinogenesis [43]. In the present study, viral load and physical state of infecting virus 9 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis LSIL (n=30) HSIL (n=30) SCC (n=70) Fig 3. Analysis of the physical state of HPV16 genome and level of active pSTAT3(Y705) in cervical pre-cancer and cancer cases. Distribution of normalized HPV16 E2: E6 ratio and level of pSTAT3 expression in cervical pre-cancer (LSIL and HSIL) and cancer cases (SCC). Each circle indicates an individual case. Normalized E2: E6 ratios were determined by PCR and calculated as described in Methods. Normalized E2: E6 ratio = 0 represents predominantly integrated HPV16 genome, value = 1 represents predominantly episomal viral genome, and values between 0 and 1 indicate a mixed Fig 3. Analysis of the physical state of HPV16 genome and level of active pSTAT3(Y705) in cervical pre-cancer and cancer cases. Distribution of normalized HPV16 E2: E6 ratio and level of pSTAT3 expression in cervical pre-cancer (LSIL and HSIL) and cancer cases (SCC). Each circle indicates an individual case. Normalized E2: E6 ratios were determined by PCR and calculated as described in Methods. Normalized E2: E6 ratio = 0 represents predominantly integrated HPV16 genome, value = 1 represents predominantly episomal viral genome, and values between 0 and 1 indicate a mixed form of HPV16 genome. 1Arbitrary level of pSTAT3 expression in immunoblotting: Strong = (+++), Moderate = (++), Weak = (+), Nil/undetectable = (-). The vertical red line and horizontal blue line represent median values of pSTAT3 expression and E2: E6 ratio in each disease group, respectively. Fig 3. Analysis of the physical state of HPV16 genome and level of active pSTAT3(Y705) in cervical pre-cancer and cancer cases. Distribution of normalized HPV16 E2: E6 ratio and level of pSTAT3 expression in cervical pre-cancer (LSIL and HSIL) and cancer cases (SCC). Each circle indicates an individual case. Normalized E2: E6 ratios were determined by PCR and calculated as described in Methods. q arson Chi-square p<0.001between two levels of STAT3 expression concerning the three different physical status of HPV16. ld type refers to statistically significant p values. https://doi.org/10.1371/journal.pone.0222089.t004 Tissues derived from Low-grade Squamous Intraepithelial Lesions; HSIL—Tissues derived from High-grade Squamous Intra from Squamous Cell Carcinoma; SE–Standard Error quamous Intraepithelial Lesions; HSIL—Tissues derived from High-grade Squamous Intraepithelial Lesions; SCC—Tissues SE–Standard Error pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis Table 5. Correlation between active pSTAT3 with physical state of HPV16 and its viral load in different disease grade of cervical cancer. Stage of Disease Active pSTAT3 Expression Level HPV16 Physical State HPV16 Viral Load (GE/Unit Host Genome) p-value Median Mean ± SE LSIL Nil/Low (n = 24) Episomal (n = 21) 25 27.3 ± 3.0 Mixed (n = 3) 45 34.7 ± 12.9 Integrated (n = 0) - - Moderate/ Strong (n = 6) Episomal (n = 6) 47.5 45.17 ± 3.7 0.012a Mixed (n = 0) - - NAb Integrated (n = 0) - - NAc HSIL Nil/Low (n = 18) Episomal (n = 11) 68 64.9 ± 6.7 Mixed (n = 5) 152 198.0 ± 45.0 Integrated (n = 2) 227 227.0 ± 38.0 Moderate/ Strong (n = 12) Episomal (n = 6) 126.5 136.1 ± 25.75 0.010a Mixed (n = 3) 1059 875.1 ± 302.9 0.071b Integrated (n = 3) 400 435.0 ± 154.8 0.800c SCC Nil/Low (n = 13) Episomal (n = 3) 155.00 278.7 ± 168.5 Mixed (n = 5) 183.00 235.4 ± 61.3 Integrated (n = 5) 245.00 251.0 ± 34.0 Moderate/ Strong (n = 57) Episomal (n = 2) 92.00 92.00 ± 24.000 0.4a Mixed (n = 24) 2637.00 3098.29 ± 484.322 <0.001b Integrated (n = 31) 559.00 843.90 ± 116.970 0.001c aepisomal vs episomal bmixed vs mixed cintegrated vs integrated between nil/weak and moderate/strong pSTAT3 expression within each disease stage; p-value determined by Mann-Whitney U test, NA- not applicable due to the zero value in particular category. https://doi.org/10.1371/journal.pone.0222089.t005 th physical state of HPV16 and its viral load in different disease grade of cervical cancer. bmixed vs mixed cintegrated vs integrated between nil/weak and moderate/strong pSTAT3 expression within each disease stage; p-value determined by Mann-Whitney U test, NA- not applicable due to the zero value in particular category. https://doi org/10 1371/journal pone 0222089 t005 mixed vs mixed integrated vs integrated between nil/weak and moderate/strong pSTAT3 expression within each disease stage; p-value determined by Mann-Whitney U test, NA- not pplicable due to the zero value in particular category. were collectively examined for any correlation with expression of pSTAT3 to assess whether these are related events during the progression of cervical carcinoma. Discussion Normalized E2: E6 ratio = 0 represents predominantly integrated HPV16 genome, value = 1 represents predominantly episomal viral genome, and values between 0 and 1 indicate a mixed form of HPV16 genome. 1Arbitrary level of pSTAT3 expression in immunoblotting: Strong = (+++), Moderate = (++), Weak = (+), Nil/undetectable = (-). The vertical red line and horizontal blue line represent median values of pSTAT3 expression and E2: E6 ratio in each disease group, respectively. https://doi.org/10.1371/journal.pone.0222089.g003 Table 4. HPV16 physical status and level of pSTAT3 expression in cervical precancer and cancer lesions. Tissue Type pSTAT3 Levels Physical State of HPV16 Genome Total (%) p-value Episomal (%) (E2:E6 1.0) Mixed (%) (0 < E2:E6 > 1.0) Integrated (%) (E2:E6 = 0) LSIL (n = 30) Nil/Weak 21 (70) 3 (10) - 24 (80) 0.361 Moderate/Strong 6 (20) - - 6 (20) HSIL Nil/Weak 11 (36) 5 (17) 2 (7) 18 (60) 0.603 (n = 30) Moderate/Strong 6 (20) 3 (10) 3 (10) 12 (40) SCC Nil/Weak 3 (4) 5 (7) 5 (7) 13 (18) <0.04 (n = 70) Moderate/Strong 2 (3) 24 (35) 31 (44) 57 (82) Total (n = 130) Nil/Weak 35 (27) 13 (10) 7 (5) 55 (42) <0.001a Moderate/Strong 14 (11) 27 (21) 34 (26) 75 (58) Table 4. HPV16 physical status and level of pSTAT3 expression in cervical precancer and cancer lesions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 10 / 16 Even though E2: E6 ratio is a gross test to evaluate integration and there are more sophisti- cated assays like real-time quantitative PCR, APOT or NGS, it is sufficiently indicative to infer a potential loss of E2 expression in the sample. Further, the E2 PCR characteristically covers full length HPV16 E2 region (amplicon size– 1139bp) which eliminates the chances of falsely interpreting integrated HPV as episomal. The full length E2 primers used and the PCR based assay to assess physical state was developed and validated by our laboratory previously [37][2]. The E2 primers produced a considerably long PCR product (1139bp) that was not amenable to Table 6. Assessment of correlation between pSTAT3 with HPV16 viral load and E2: E6 Ratio in HPV16-positive cervical tissues (n = 130). Variables Viral Load E2: E6 Ratio pSTAT3 Lesion grade ρ 0.810 -0.710 0.518 p-value 0.0000002 0.0000002 0.000000000215 Viral Load ρ -0.691 0.696 p-value 0.0000002 0.0000002 E2:E6Ratio ρ -0.474 p-value 0.0000000163 ρ- Spearman’s rank-order correlation coefficient https://doi.org/10.1371/journal.pone.0222089.t006 PLOS ONE \| https://doi org/10 1371/journal pone 0222089 September 5 2019 11 / 16 AT3 with HPV16 viral load and E2: E6 Ratio in HPV16-positive cervical tissues (n = 130). correlation between pSTAT3 with HPV16 viral load and E2: E6 Ratio in HPV16-positive cervical tissues (n = 130). Assessment of correlation between pSTAT3 with HPV16 viral load and E2: E6 Ratio in HPV16-positive cervical tissues PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 11 / 16 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis Table 7. Pairwise multiple comparison by tukey test between different study variables. Comparison Difference of Ranks q score p-value Viral Load vs. E2:E6 Ratio 49304.5 28.780 <0.001 Viral Load vs. Lesion grade 28076.5 16.389 <0.001 Viral Load vs. pSTAT3 23925.0 13.965 <0.001 pSTAT3 vs. E2:E6 Ratio 25379.5 14.814 <0.001 pSTAT3 vs. Lesion grade 4151.5 2.423 0.317 Lesion grade vs. E2:E6 Ratio 21228.0 12.391 <0.001 https://doi.org/10.1371/journal.pone.0222089.t007 Table 7. Pairwise multiple comparison by tukey test between different study variables. real-time quantitation. This restricted us to stay with the conventional end-point PCR tech- nique. The gross E2: E6 ratios derived from densitometric values of each samples post PCR were normalized to E2: E6 ratio obtained from densitometric values of concurrently amplified HPV16 plasmid which was taken as control and represented the episomal state of the HPV genome. The present study specifically focused on HPV16 genotype. Viral load of HPV16 but not the HPV type 18 was demonstrated to express the highest predicted value (96% specificity and 88% sensitivity) in an earlier study [44]. In a similar study on HPV16, HPV18, HPV31, and HPV45, a clear dose-response pattern relationship between HPV16 DNA loads and infection clearance was observed, whereas, other types failed to show such relationship [45]. Therefore, in the present study, tissues positive for other types of HPV infections, with or without HPV16 infection, that could potentially introduce confounding variables were excluded from the anal- ysis to maintain the homogeneity of the study. The present investigation revealed that expression of pSTAT3(Y705) within different stages of the cervical carcinogenesis (LSIL, HSIL or SCC) individually as well as collectively, was asso- ciated with the degree of HPV16 viral load. A recent study has experimentally shown that loss of STAT3 severely impacts episomal maintenance of HPV18 in undifferentiated keratinocytes [36]. The study highlighted the role of viral oncoprotein E6 in HPV18 viral genome amplifica- tion, whereas, a high level of E6 was reported in invasive cervical cancers that had episomal HPV16 [46]. In an analogous system of virally-induced hepatocellular carcinoma, the role of active STAT3 has been suggested in HCV replication [47]. STAT3-mediated molecular mecha- nisms that operate and control HPV16 viral load in infected lesions are, however, unknown. Earlier studies from our laboratory showed expression of HPV16 E6 forms a positive regula- tory loop with STAT3 [43,48] and other stemness-associated markers, GLI and Hes1, to pro- mote oncogenic activity [34,49]. This could be likely via selective overexpression of E6 in some of the cells positive for HPV16, if not all cells of the lesions, which could lead to increase pSTAT3 level in patients with severe disease (and integrated genomes). HPV E6 has been implicated in activation of EGFR and expression of IL-6 and Oncostatin M [36,50–52], which collectively work as STAT3 upstream activators. However, any direct action of increased pSTAT3 on the integration of HPV16 in the host genome in either cancer or precancer tissues, is not known as yet. The present investigation only provides evidence of a positive correlation between these two events, and is suggestive of a potential interlink. Due to the lack of an appropriate experimental model for HPV16 integration, the possibility of evaluating the direct influence of active pSTAT3 on HPV16 integration events or vice versa remains unexplored. Acknowledgments We acknowledge Dr. Swaraj Batra for helping in the collection of clinical specimen and Dr. Suresh Bhambhani for evaluation of tumor tissues and pathological grading during the study. We are thankful to Dr. Archana Kumari, Amity Institute of Biotechnology, NOIDA, U.P for assisting in statistical evaluation of the results. We acknowledge Prof. Sukh Mahendra Singh, Bananas Hindu University, Varanasi for critically reviewing, proofreading and assisting in sci- entific English writing. Author Contributions Conceptualization: Shirish Shukla, Alok Chandra Bharti. Conceptualization: Shirish Shukla, Alok Chandra Bharti. Data curation: Shirish Shukla, Gauri Shishodia, Sutapa Mahata. Data curation: Shirish Shukla, Gauri Shishodia, Sutapa Mahata. Formal analysis: Shirish Shukla, Mohit Jadli, Kulbhushan Thakur, Gauri Shishodia, Sutapa Mahata, Bhudev Chandra Das, Alok Chandra Bharti. Formal analysis: Shirish Shukla, Mohit Jadli, Kulbhushan Thakur, Gauri Shishodia, Sutapa Mahata, Bhudev Chandra Das, Alok Chandra Bharti. Funding acquisition: Alok Chandra Bharti. Funding acquisition: Alok Chandra Bharti. Investigation: Shirish Shukla, Gauri Shishodia, Sutapa Mahata, Alok Chandra Bharti. Methodology: Shirish Shukla, Sutapa Mahata, Alok Chandra Bharti. Project administration: Alok Chandra Bharti. Resources: Alok Chandra Bharti. Supervision: Seemi Farhat Basir, Bhudev Chandra Das, Alok Chandra Bharti. Validation: Alok Chandra Bharti. Writing – original draft: Mohit Jadli, Kulbhushan Thakur, Bhudev Chandra Das, Alok Chan- dra Bharti. Writing – review & editing: Seemi Farhat Basir, Bhudev Chandra Das, Alok Chandra Bharti. Given these observations, our data provide first clinical evidence that this phenomenon could also operate in an in vivo situation in HPV16-infected cervical precancer and cancer lesions. A comparative analysis of pSTAT3 expression with HPV16 physical state further demon- strated that in most of the LSIL cases where the HPV16 genome was present in predominantly episomal form, STAT3 activation was weak. On the other hand, tissues of HSIL and cancer 12 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222089 September 5, 2019 pSTAT3 correlates with HPV16 viral load and physical state in cervical carcinogenesis lesion harboring either mixed or predominantly integrated form of the HPV16 genome showed an elevated level of active pSTAT3. This correlation sustained in cancer tissues harbor- ing episomal form demonstrated a low level of active STAT3. Our data, therefore, indicated a possible association between STAT3 activation and integration of the viral genome in HPV16 infections. The mechanism by which STAT3 mediates its effect on the process of integration, which is considered as an opportunistic and random event during carcinogenesis, is not known. In such a scenario, an indirect mechanism, such as the induction of genomic instabil- ity [26] by which STAT3 could influence integration events, cannot be ruled out. k h d h f h f l l l b Taken together, our study shows for the first time a clinical correlation between pSTAT3, viral copy number, and physical state of the HPV16 genome during cervical disease progres- sion. Independent of its direct role in epithelial carcinogenesis [31], STAT3 may be involved in existence as well as pathological manifestation of HPV16 genome and could be utilized as pos- sible therapeutic, diagnostic and prognostic target for potentially-progressive cervical cancer and pre-cancer lesions. References 1. Zur Hausen H, Rosl F (1994) Pathogenesis of cancer of the cervix. 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W2320353702.txt	null	en		Flux and accumulation of sedimentary particles off the continental slope of Pakistan: a comparison of water column and seafloor estimates from the oxygen minimum zone, NE Arabian Sea	null	2,013	cc-by	12,255	Biogeosciences, 11, 3107–3120, 2014 www.biogeosciences.net/11/3107/2014/ doi:10.5194/bg-11-3107-2014 © Author(s) 2014. CC Attribution 3.0 License. Vertical and lateral flux on the continental slope off Pakistan: correlation of sediment core and trap results H. Schulz1,* and U. von Rad2,* 1 Fachbereich für Geowissenschaften, Paleobiology, University of Tübingen, Hölderlinstr. 12, 72074 Tübingen, Germany 19c, 30916 Isernhagen, Germany * formerly at: Bundesanstalt für Geowissenschaften und Rohstoffe (BGR), PF 510153, 30631 Hannover, Germany 2 Rosenstraße Correspondence to: H. Schulz (hartmut.schulz@uni-tuebingen.de) Received: 13 May 2013 – Published in Biogeosciences Discuss.: 23 July 2013 Revised: 27 March 2014 – Accepted: 12 April 2014 – Published: 16 June 2014 Abstract. Due to the lack of bioturbation, the varvelaminated muds from the oxygen minimum zone (OMZ) off Pakistan provide a unique opportunity to precisely determine the vertical and lateral sediment fluxes in the nearshore part of the northeastern Arabian Sea. West of Karachi (Hab area), the results of two sediment trap stations (EPT and WPT) were correlated with 16 short sediment cores on a depth transect crossing the OMZ. The top of a distinct, either reddishor light-gray silt layer, 210 Pb-dated as AD 1905 ± 10, was used as an isochronous stratigraphic marker bed to calculate sediment accumulation rates. In one core, the red and gray layer were separated by a few (5–10) thin laminae. According to our varve model, this contributes < 10 years to the dating uncertainty, assuming that the different layers are almost synchronous. We directly compared the accumulation rates with the flux rates from the sediment traps that collected the settling material within the water column above. All traps on the steep Makran continental slope show exceptionally high, pulsed winter fluxes of up to 5000 mg m−2 d−1 . Based on core results, the flux at the seafloor amounts to 4000 mg m−2 d−1 and agrees remarkably well with the bulk winter flux of material, as well as with the flux of the individual bulk components of organic carbon, calcium carbonate and opal. However, due to the extreme mass of remobilized matter, the high winter flux events exceeded the capacity of the shallow traps. Based on our comparisons, we argue that high-flux events must occur regularly during winter within the upper OMZ off Pakistan to explain the high accumulations rates. These show distribution patterns that are a negative function of water depth and distance from the shelf. Some of the sediment fractions show marked shifts in ac- cumulation rates near the lower boundary of the OMZ. For instance, the flux of benthic foraminifera is lowered but stable below ∼ 1200–1300 m. However, flux and sedimentation in the upper eastern Makran area are dominated by the large amount of laterally advected fine-grained material and by the pulsed nature of the resuspension events at the upper margin during winter. 1 Introduction About 1 million km3 of shelf and slope waters of the global ocean are found to be permanently hypoxic, with more than half of that volume in the Indian Ocean (Helly and Levin, 2004). After the pioneering study of von Stackelberg (1972) on the sedimentation along the Indian–Pakistan margin of the northern Arabian Sea, numerous campaigns and initiatives revealed the key role of the organic-rich, laminated sediments in studying the linkages between the production, pathways and preservation of marine organic matter under low bottomwater oxygen conditions (Paropkari et al., 1992; Pedersen et al., 1992; Calvert et al., 1995; Cowie et al., 1999; Keil and Cowie, 1999; van der Weijden et al., 1999; von Rad et al., 1999; Schulte et al., 2000; Suthhof et al., 2000; Cowie, 2005; Wiggert et al., 2005; Cowie and Levin, 2009). The northern Arabian Sea off Pakistan is characterized by a stable, distinct oxygen minimum zone (OMZ) between water depths of 200 m and about 1200 m that impinges on the continental slope. The very low oxygen concentrations at intermediate depths (< 0.2 mL L−1 ) result from low ventilation and from oxygen consumption by microbial Published by Copernicus Publications on behalf of the European Geosciences Union. EPT 0 500 1000 1500 2000 2500 3000 C WPT 641586644 42 69 35 2 3 4 155 5 639 58 53 142 76 1 72 143 46 shallow OMZ 66°E ol Hing r Rive 142 158 1 58 53 69 64 76 143 39 35 deep 14479 54 85 144 79 R.r 64°E us Ba s ul 85 54 Ind Makran sal bys in Pla nA a Om 67°E R. m w.d. H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan Por ali 3108 A er EPT 6 54 3 2 72 42 4466 51 46 WPT b Ha v Ri 25°N KARACHI B 24°N Figure 1. Investigation area in the northwestern Indian Ocean off Pakistan. (A) Arabian Sea with Makran and Indus continental margins. (B) Position of sediment trap deployments and sediment stations (“Hab area” W of the Hab river) from two oceanographic cruises, SONNE 90 (numbers 35 onward) and METEOR 32/2 (numbers 1–6). Most sites are distributed below the narrow Makran shelf to the northeast and some down the broader Indus margin to the southeast. (C) Idealized transect “Hab transect” of sediment stations between 92 and 2881 m with sediment traps EPT and WPT. Distance between traps is ∼ 20 km. Stippled area is the oxygen minimum zone, OMZ. decomposition of organic matter within the water column and on the seafloor. High surface water primary productivity and organic matter flux is triggered by the strength and direction of the seasonally reversing summer (southwest) and winter (northeast) monsoonal circulation, and may also depend on fluvial and eolian terrigenous input (Sirocko and Sarnthein, 1989; Ramaswamy et al., 1991). Vertical fluxes in the open oceanic environment in the western Arabian Sea were intensely studied by means of time series of moored and drifting sediment traps (Haake et al, 1993; Nair et al., 1989; Rixen et al., 1999; Pollehne et al., 1993). In contrast, our knowledge on the scales and seasonal timings of the marine biogenic and terrigenous fluxes on the steep margin zones of the northern Arabian Sea is still fragmentary. The sediments along the continental slope off Pakistan (Fig. 1a) are characterized by a distinct lamination due to suppressed bioturbation (von Stackelberg, 1972). However, the hypoxic bottom water conditions preventing the sediment from being mixed by burrowing organisms may be only one important factor for the preservation of laminae. Exceptionally high sedimentation of more than 1 mm per year, linked to the high lateral fluxes of remobilized terrigenous and marine matter, was observed off the active, steep Makran margin off western Pakistan (Schulz et al., 1996; Andruleit et al., 2000; Lückge et al., 2002; Schulz et al., 2002; von Rad et al., 2002a, b). Thus, high fluxes and sediment redistribution may also contribute to the preservation of sediment lamination. However, comparable laminations are not observed off India, where slope sedimentation rates can be as high, nor are they off Oman, where slopes are also steep (Paropkari et al., Biogeosciences, 11, 3107–3120, 2014 1992). But sediment laminations are also found off the Indus Delta, where sedimentation rates are lower and the slope is less steep (Cowie et al., 1999). From sedimentary models of varve formation and from simple mass calculations, we may expect a significant contribution of fine-grained, remobilized matter to the steep continental slope from the narrow shelf areas nearby. This adds also to the particle flux derived from local biological productivity and thus complicates the identification of the sediment sources and processes. Also, MODIS satellite images show examples of distinct sediment plumes extending from the coastal zone offshore (Bourget et al., 2010). These plumes might also contribute to the formation of the winter sediment laminae. The cores from the steep Hab area west of Karachi (Fig. 1b) host the most distinct sediment laminae on millimeter to sub-millimeter scales (von Rad et al., 1999; 2002b). The distinct nature of the varved sediments along the steep Makran is dominated by a high fraction of lithic components. For at least the past 5000 years, there has been a continuous deposition of annual laminae couplets, which point to a strong seasonality in the amount and composition of material settling to the seafloor (von Rad et al., 1999). Microscopic analyses of thin sections show distinct alternations of dark-colored, organic-carbon-rich and light-colored, organic-carbon-poor terrigenous laminae, which suggest a cyclic pattern due to changes in the flux and composition during summer and winter (Berger and von Rad, 2002; Lückge et al., 2002). Cores from the less steep Indus Pakistan margin, situated to the southeast and distant from the Makran, are characterized by a more regular pattern in lamina thickness. Quantitative flux estimates are needed to better describe the complex sedimentation processes in the water column and on the seafloor at the continental margin off Pakistan. However, direct observations of the summer and winter situations are missing to corroborate the formation of the sediment laminae and the transfer of sediments to the deep sea. Four sediment trap moorings in eastern Pakistan (EPT) and western Pakistan (WPT) (Fig. 1) have been deployed within and below the OMZ off Pakistan. However, the flux estimates from these traps were only fragmentary and showed strong, unexpected temporal and spatial variability. None of the four traps in 1993/1994 and 1995/1996 sampled the full year as planned. All traps stopped collecting in the winter season, three of four traps presumably due to a huge flux of matter remobilized on the upper margin (von Rad et al., 1995; Andruleit et al., 2000; Schulz et al., 2002; von Rad et al., 2002b). The reason for these flux events recorded in the traps and their role in the sedimentation off Pakistan is poorly constrained. In the present study, we focus on the quantification of sediments accumulated along a depth transect in the Hab area (Fig. 1) between ∼ 200 and ∼ 2000 m water depth, and directly compare these accumulation rates with the limited numbers of flux estimates derived from sediment traps deployed in the overlying water column. Published estimates of www.biogeosciences.net/11/3107/2014/ H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan SO90-39KG (24°50.0’N, 65°55.0’E, 704m w.d.) a) Dry Bulk Density (g cm-3) 0.80 1.00 0.40 0.60 www.biogeosciences.net/11/3107/2014/ 2000 1900 1800 1700 15 20 25 30 35 40 45 M32-2MC5 (24°38.4’N 66°02.9’E, 386m w.d.) b) Sediment Lightness Lab-Scale 40 45 50 0 Sand Fraction (%) 1 2 3 4 Dry Bulk Density (g cm-3) 0.4 0.6 0.8 1.0 1.2 10 OC (%) CaCO3 (%) 15 20 0 1 2 3 >1950 AD Core depth (cm) 5 10 15 664+28 20 25 705+27 30 laminated c) turbidite M32-2MC3 23°37.9’N 66°04.6’E, 247m w.d.) 40 Sediment Lightness Lab-Scale 45 50 0 1 Sand Fraction (%) 2 3 4 Dry Bulk Density (g cm-3) 0.4 0.6 0.8 1.0 1.2 10 CaCO3 (%) 20 15 OC (%) 25 0 1 2 3 0 Core depth (cm) 5 10 15 20 25 30 SO90-58KG (24°46.6’N 65°49.2’E, 876m w.d.) d) Dry Bulk Density (g cm-3) 0.2 0.4 0.6 0.8 Sediment Color/Red-Channel Lab-Scale 1.0 -2.0 -1.0 0.0 Varve Thickness (mm) 1.0 10.0 Age (Varve Years A.D.) 2000 1950 1900 0 5 Core depth (cm) The study area on the continental margin of Pakistan in the northeastern Arabian Sea represents a restricted area between the narrow active Makran margin to the northwest and the Indus margin to the southeast (Fig. 1a), where the broad shelf is typical for the passive Indian margin (Fig. 1a). The “Hab area” (Fig. 1b) is characterized by the three tectono-sedimentologic settings: (1) to the north the accretionary complex covered by thick uplifted wedges and sediment prisms that is part of the steep Makran margin (stations 39, 53), (2) a lower accretionary wedge zone with rather regular topography on vertical scales of some 100 m (stations 1, 35, 58–158) and (3) a more gentle NE slope of the Murray Ridge to the southeast that was formed by large blocks of slumped sediments (stations 2–6, 42–46, 54). The Hab area is drained by deep, mostly ENE–WSW-trending channels. In the near-surface sections of 16 box cores and 6 multicores, up to 12 cm thick reddish- or gray-whitish-colored silty-clay layers were identified (Staubwasser and Sirocko, 2001). Based on the strong visual contrast of both layer types to the over- and underlying laminated sediment (Fig. 2), these thick layers can be traced over large overlapping areas, possibly covering some 1000 and 600 km2 , respectively. Most cores show either the red or the gray-whitish layer. The red layer is found more on the Makran side, to the north of the ENE–WSW-trending channels. All laminated SONNE cores (except station 35 at 416 m) showed a distinct reddishcolored silt layer that can be regarded as a silt turbidite, sometimes with fining upward or indistinct internal laminations (Schulz et al., 1996). In contrast, the gray-whitish layer is observed more to the southeast on the Indus side, at shallower depths (stations 2–6; see also supplementary online material), but not at all sites: cores 44 KG and 42 KG are more similar to the Makran cores (Staubwasser and Sirocko, 2001). In core 58 KG (Fig. 2d) presumably both types of layers were found, with the reddish-colored layer (∼ 13–22 cm) above the whitish one (∼ 22.5–25.5 cm). The whitish layer is separated from the red layer by 5–10 thin sediment laminae. The two types of fine-grained clastic sediment appear to be Age (Varve Years A.D.) Varve Thickness (mm) 1.0 10.0 100.0 5 0 Geologic setting and previous geological investigations 0.1 10 35 2 1.20 0 Core depth (cm) surface water productivity can be used to quantitatively better describe the local flux of organic carbon at a certain depth derived from primary production (Sarnthein et al., 1992). However, this approach may not be valid in the near-coastal environment, where lateral fluxes are high and may be temporally dominant. To better determine their role, we will focus on possible differences in the amount and preservation of bulk fractions of biogenic and mineral matter found above, in the center and below the OMZ. The concentration and accumulation of the major sand-sized components preserved along our core transect across the OMZ will be used to provide additional information. 3109 10 15 20 25 30 2000 1950 1900 Age (Years AD) Pb-Dating laminated turbidite bioturbated Figure 2. Examples from four short sediment cores along the Pakistan margin for high-resolution analyses of sediment properties and dating. Various properties display marked changes because of a 4 to 10 cm thick reddish-gray turbidite deposit. Profiles from the OMZ show distinct sediment laminations as well. According to models of their formation, varve counts, 210 Pb- and 14 C dating provide a coherent age of AD 1905–1888 for that layer. Biogeosciences, 11, 3107–3120, 2014 3110 H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan very similar in their elemental ratios, as well as in their bulk organic carbon and calcium carbonate contents (Staubwasser and Sirocko, 2001, unpublished data). The type of reddishcolored sediment also occurs in up to a few centimeter-thick intervals in the longer piston and long box cores of the area, interpreted to represent an episodic redeposition of fluvial matter from the Indus shelf, possibly linked to strong rainfall or floods in the hinterland (Schulz et al., 1996; von Rad et al., 2002a). Staubwasser and Sirocko (2001) investigated the light-gray clastic sediment in more detail and concluded that these might consist of material expelled by mud volcanoes from the active Makran margin. In contrast von Rad et al. (2002a) argued that the distinct light-gray to whitish layers are explained as “plume deposits” transporting mudcharged suspensions across the narrow shelf to the slope. No additional, similarly thick layers are observed further downcore in the longer Holocene records from the Hab area. By establishing the stratigraphic position of the turbidite and plume sedimentation events, the unique event layers, interbedded between laminated sediment, could be ideally used as stratigraphic marker beds for correlation of the short cores. The described layers were sampled again by SONNE cruise 130 in the Hab area by the following box cores (KG) and multicores (MC): 268 MC, 605 m; 276 KG, 780 m; 262 MC, 875 m; and 291 MC, 902 m water depth. In cores 262 MC and 291 MC from the same area as 58 KG, the red and whitish layers were found on top of each other again, questioning their exact synchronism. 3 3.1 Material and methods Sediments and accumulation rates Twenty box cores (KG) and 6 multicores (MC) (short gravity cores of ∼ 10–50 cm length) were taken in 1993 during SONNE cruise SO 90 and in 1995 during METEOR cruise M32/2, respectively, and recovered undisturbed top sediments from water depths between 96 and 2881 m. The organic carbon (OC), calcium carbonate (CaCO3 ) and opal concentrations were determined following Dietrich and Marchig (1995) and Suthhof et al. (2000). The composition of the coarse fraction larger than 150 microns (µm) in core top samples was counted from a few milliliters of freeze-dried sediment of the top centimeter washed through a 63 µm mesh. The residue was dried at 60 ◦ C and sieved into sub-fractions of 63–150, 150–200, 200–250, 250–315, 315– 400 and > 400 µm, respectively. Splits of at least 70 grains from the fractions > 150 µm were obtained with an Otto microsplitter for each sample. In these splits all grains were identified following the categories (1) inorganic compounds (Inorg.: quartz, mica, oxides, lithic grains), (2) megafossils (Megaf.: sponge spicules, ophiurid remains, pteropods, fish debris, etc.), (3) siliceous tests (Silic.: large radiolarians, diatoms, silicoflagellates), (4) planktic foraminiferal debris Biogeosciences, 11, 3107–3120, 2014 (P.F. debris), (5) intact planktic foraminiferal tests (P.F.) and (6) benthic foraminifera (B.F.). The six categories represent more than 95 % of the > 150 µm size fraction. They were used to better characterize the texture and composition of the surface sediment and to identify possible remobilization of sand-sized particles across the steep margin. For example, potential disturbances of the fluffy top layer could be deduced from coarse fractions. Increased numbers of the lithic fractions would indicate mass flows or sediment winnowing. Further, calcite dissolution or corrosion at certain depth may be identified by an increased fragmentation of planktic foraminiferal shells. Profiles of sediment γ -ray attenuation were analyzed at 0.5 cm intervals using a GEOTEC-multisensor core logger (Schultheiß and Weaver, 1992). Discrete samples were taken at 1–2.5 cm intervals by 10 cm3 syringes or from sliced sediment profiles in order to determine the sediment physical properties of natural water content as well as wet and dry bulk density. The latter two were used to calibrate the parallel profiles of γ -ray attenuation that vary as a function of sediment density (Weber et al., 1997). Using these calibrations from selected cores, a homogenous set of dry bulk density data was generated for 15 sediment profiles of 12 to 40 cm length from water depths of 250 to 1970 m. The 30 cm long multicore MC2 from a depth of 197 m was only described visually prior to discrete sampling, and showed a homogenous light-colored layer of silty clay between 18.0 and 21.0 cm core depth. A detailed varve chronology was developed for the laminated sediments from the Pakistan margin (Schulz et al., 1996; von Rad et al., 1999). This chronology is based on the assumption that a couplet of one light-gray-colored (winter) lamina plus one dark-gray, organic-rich (summer) lamina was deposited in one year. This assumption was verified by the observation that five light–dark couplets of laminae were accumulated at the upper Makran slope in between 1993 (SONNE 90) and 1998 SONNE 130). Lückge et al. (2002) interpreted the thicker light-colored laminae as representing recurrent suspension events linked to high winter precipitation, and the dark-colored laminae as caused by the high summer productivity during the SW monsoon. About 150 samples from the top 20 cm of laminated sediment at 1 cm intervals of cores MC1, MC3, MC5 and 39 KG, 58 KG were used for 210 Pb dating to precisely determine the sedimentation rates during the last 150 years. These data are presented here only for core 58 KG (Erlenkeuser, unpublished data). The relative error for the past 1000 years is better than 10 % for that method. Analytical details of the 210 Pb method are outlined in Erlenkeuser and Peterstad (1984). AMS14 C ages of monospecific samples of Globigerinoides ruber (white) or of mixed P.F. were determined at the Leibniz Labor Kiel, Germany. In order to obtain a quantitative measure on the flux of individual particles and compounds to the seafloor, accumulation rates (ARs) were determined for the 16 short cores, www.biogeosciences.net/11/3107/2014/ H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan based on the sedimentation rate estimates from the cores and from the dry bulk densities, following Thiede et al. (1982): ARbulk sediment = SR pdry , (1) with AR in g cm−2 kyr−1 , SR in cm kyr−1 , and pdry (dry bulk density) in g cm−3 . The accumulation rate of individual compounds, or of the individual sediment components in a sample, is calculated as follows: ARcomponent = SR pdry C/100, (2) where C is the concentration of a compound in percent, and N is the number of the specimens per gram of dry sediment. 3.2 Sediment traps Four trap moorings of type MARK 7G-21 (McLane Research Laboratories Inc.) at sites EPT (24◦ 45.40 N, 65◦ 48.70 E) and WPT (24◦ 35.90 N, 65◦ 35.30 E; 2004 m seafloor depth) had been deployed within and below the OMZ off Pakistan (Fig. 1). Collection time was programmed as October 1993 to February 1994 and May 1995 to February 1996 (EPT2). Sampling intervals for the 28 cups used in the present study were 22 days for WPT and EPT1 and 24 days for EPT2, respectively (Table 2). Prior to deployment, cups were treated with mercuric chloride. The total mass fluxes, CaCO3 and lithogenic particle fluxes were also presented in Andruleit et al. (2000) and in Schulz et al. (2002), who studied the coccolithophore and planktic foraminiferal fluxes, and by Treppke, who studied the diatoms (von Rad et al., 2002b, their Fig. 11). Details of trap and sample preparation, OC, CaCO3 , biogenic opal analyses, and of the derivation of fluxes are described in Haake et al. (1993), Andruleit et al. (2000) and Suthhof et al. (2000). The mass accumulation rates were previously used by Suthhof et al. (2000). All flux and accumulation rate results are listed in Table 2. Estimates of local primary productivity were adopted from Antoine at al. (1996). The accumulation rate for the respective sediment property was derived from that analysis of the core top sample 0–1 cm. The density value for a particular station was averaged from the measurements above the marker layer (Fig. 3). Similarly, sedimentation rates were calculated assuming a linear accumulation process of the laminated/bioturbated sediments over the past ∼ 100 years. Most estimates for the study are expressed as mg m−2 d−1 or specimens (= spec.) m−2 d−1 to allow for direct comparison. 4 4.1 Results A turbidite or turbid top layer as stratigraphic marker for core correlation Staubwasser and Sirocko (2001) presented sedimentological and geochemical data on the distinct, up to 12 cm thick reddish turbidite they observed in nearly all SONNE cores of the www.biogeosciences.net/11/3107/2014/ 3111 Hab area. An investigation of the METEOR short multicores, overlapping with the SONNE sediments, shows in some cases that METEOR cores 1–6 are thinner, having a gray to lightgray silty layer at 10 to 24 cm depth (see supplementary online material). The cores 2–6 were taken from a channel, while cores 42 and 44 KG ware recovered from the nearby flank of that channel on the NE slope of the Murray Ridge, (Fig. 1b). We show that both types of layers display internal structures that can be best seen in their high-resolution profiles of dry bulk density (Fig. 2b, d). We observe faint grading in the reddish-colored, decimeter-thick beds in some SONNE cores, as well as highest density at their bottoms (Fig. 3), that might reflect coarser grain sizes or compaction at the base of these layers. The density profile of shallow core 44 KG suggests that the thick layers might also display more internal structure reflecting at least two sublayers. From a slight increase in sand content near the base, faint regular grading can also be traced in the relatively thin, light-gray layer of core MC5 (Fig. 2b). All cores display high sediment densities for the layer of ≥ 0.8 (Fig. 3) corresponding to low OC content of < 1 % (Fig. 2). Few pelagic microfossils, grading and rapid flow expansion leading to variable thicknesses are typical diagnostic features of that type of fine-grained silt turbidite (Piper and Stow, 1991). However, quality of the extremely soft METEOR cores is poor compared to the SONNE box cores, where most of these criteria can be observed. Due to increasing bottom water oxygen at depths above and below the OMZ supporting macrofaunal bioturbation, the marker could not be determined with precision outside the OMZ. The upper boundary of that marker layer is still feasible as a slight relative increase in density in the high-resolution logs below the OMZ down to 1970 m (Fig. 3). Although the sedimentation at the central Makran is characterized by the deposition of up to some millimeter- to decimeter-thick lightcolored “turbid layers” (von Stackelberg, 1972) and by larger turbidites (Schulz et al., 1996, von Rad et al., 2002b; Bourget et al., 2010), no other thick red and/or light-gray whitish layer was found in the upper meters of cores in the Hab area. Assuming that the reddish and light-gray to whitish layers reflect different sedimentation events, we are able to estimate the level of uncertainty from assuming that the layers are diachronous. In 58 KG (and also in cores SONNE 130– 262 MC and 291 MC), the reddish turbidite is separated from that whitish layer immediately below by thin, alternating black and olive-gray laminae. According to our varve model, these 5–10 millimeter-thick layers would correspond to less than 10 years (Fig. 2d; see X-ray figure of Staubwasser and Sirocko, 2001). 4.2 Local sedimentation rates by Pb dating, varve chronology and 14 C ages Counting of the varves in various cores, including replicate runs, yielded an age of AD 1888 and AD 1905 in 39 KG Biogeosciences, 11, 3107–3120, 2014 3112 H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan Dry bulk density (g cm-3) 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0 Core depth (cm) 10 20 30 40 MC3 247m 0.4 0.6 0.8 1.0 44KG MC6 MC5 42KG 258m 543m 386m 313m Dry bulk density (g cm-3) 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 53KG 825m 39KG 704m 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 0 ? 10 ? 20 30 40 58KG 876m 76KG 1186m MC1 1256m 143KG 1281m 72KG 1403m 46KG 1657m 144KG 1943m 79KG 1970m Figure 3. Correlation of short box (KG) and multicores (MC) by means of their sediment density (here expressed as dry bulk density) profiles above and below the silt turbidite, used for correlation (stippled line). A sudden increase in density with depth (in centimeters) is evident for the laminated cores within the OMZ. Density is lowest near the core top and increases with depth by compaction. Below OMZ depths, this boundary is smoothed by bioturbation, becoming more faint with water depth and distance from the shelf. and 58 KG, respectively (Fig. 2a, d).210 Pb dating of core 58 KG and of other short sediment profiles indicates that the younger date of AD 1905 (i.e., about 90 years before 1993/1996) may be more reliable. The error of that data amounts to not more than 10 %, which conforms to < ± 10 years. The sedimentation rate (SR) has a strong impact on the respective accumulation rates (ARs) (see Eq. 1), as changes in dry bulk density are relatively small, and in general show similar trends with very low values near the core top. The 13 density profiles show a rather uniform density maximum for the marker turbidite used for the correlation. Density profiles from above that layer show values increasing with water depth. Most profiles for the past ∼ 100–500 years from OMZ depths show values of < 0.6 g cm−3 with a major shift at ∼ 1200 m (compare 76 KG and MC1, Fig. 3) to densities of > 0.7 g cm−3 . The date of AD 1905 suggests sedimentation rates of 167 cm kyr−1 and 150 cm kyr−1 for the two core profiles, whereas by the correlation via the marker horizon, sedimentation rates at shallow depth are distinctly higher (up to 272 cm kyr−1 ). At greater depth and more distant from the coast, sedimentation rates were lower (46 KG and 144 KG, 44 cm kyr−1 ) (Table 2). Sedimentation rates of ≥ ∼ 200 cm kyr−1 on the shelf (MC2, MC3) are in line with Limmer et al. (2012), who determined a sedimentation rate of 195 cm kyr−1 in the core Indus-10 on the inner shelf at 71 m, to the east of the Hab area. In order to support our stratigraphic models, the AMS 14 C ages from intervals from above and further below the clay-silt layer in cores M32– MC1 and M32–MC5 show a difference of 40 14 C years over Biogeosciences, 11, 3107–3120, 2014 the interval of 7.3 cm in the latter core. The suggested sedimentation rate for that interval is ∼ 180 cm kyr−1 . Further, the ages of the laminated intervals can be used to assess the radiocarbon age of the local surface water. This “reservoir effect” of the ocean water is known to be highly variable in space and time. As expected, the planktic foraminiferal 14 C dates of the top sediments of MC1 and MC5 yielded negative ages caused by the post-bomb high atmospheric radiocarbon concentrations (Table 1). In contrast, the three laminated intervals in cores MC1 and MC5 from the OMZ yielded uncorrected, “conventional” 14 C ages of 747 ± 27, 664 ± 25, and 705 ± 23 years, respectively, that correspond to varve ages of AD 1820, AD 1903, and AD 1867. From the difference between the varve ages of 175 years, 92 years and 128 years of these depth intervals (which clearly predate the spike of “bomb” radiocarbon in 1964), and the corresponding “conventional” 14 C ages, we could determine essentially uniform reservoir ages of 582, 572 and 577 years from the three different 14 C dates. The average reservoir age of 577 years is different from the 640 years used by von Rad et al. (1999). 4.3 Sedimentation patterns 4.3.1 Bulk and sand fraction analysis across the OMZ The sediments on the continental margin off the coast of Pakistan can be described as organic-rich, carbonaceous silty clays with typically 1–4 % OC content and 15–30 % CaCO3 , with deposits on the Makran slope having slightly lower OC of 1–2.5 % and higher CaCO3 of ∼ 20 % on average than at www.biogeosciences.net/11/3107/2014/ H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan 3113 Table 1. List of AMS 14 C dates from latest Holocene laminated sediments of the oxygen minimum zone (OMZ) off Pakistan. Lab number Sample KIA201 KIA202 KIA773 KIA774 KIA775 M32-2MC1, 0–5 cm M32-2MC1, 18.5–22.5 cm M32-2MC5, 0–2.5 cm M32-2MC5, 15–18.5 cm M32-2 MC5, 26-28 cm Corrected pMC 100.42 ± 0.33 91.12 ± 0.31 105.05 ± 0.41 92.06 ± 0.32 91.59 ± 0.30 Conventional age (yr) > AD 1950 747 + 27 / −27 BP > AD 1950 664 + 28 / −28 BP 705 + 27 / −27 BP Varve age (yr) Reservoir age (yr) 1970 1820 1984 1903 1867 – 582 – 572 577 assuming constant sedimentation rates (Table 2). comparable depth on the Indus slope. An exception is the carbonate-rich facies of relict sands on the shelf, which contain a significant component of biogenic calcite and a bulk carbonate content of up to 70 % (von Stackelberg, 1972). These carbonate sands are restricted to shallow water depths of < 200 m on the Makran side, but are found down to 400 m below present sea level on the Indus side. The shallow sites (Fig. 1c) are situated within a relatively flat channel morphology. Above the OMZ the flat shelf may provide space and substrate for sessile and vagile CaCO3 -producing organisms, which is in line with the high relative abundance and AR of megafossils at < 250 m water depth (Fig. 4). Below that depth, the sand fraction decreases to only 0.5–3.0 wt% (Fig. 2), where most of the coarse fraction is of planktic and benthic foraminiferal tests with a few hundred to a few thousand spec. g−1 . Remarkable for OMZ sediments is the high abundance of benthic foraminifers (Fig. 4). Based on accumulation rates, we can say that the benthic foraminiferal population at the lower OMZ boundary and below shows stable but low numbers (∼ 10–50 spec. m−2 yr−1 ), while benthic foraminifera clearly are more important within the OMZ (∼ 500–1000 spec. m−2 yr−1 ). The accumulation of benthic foraminiferal shells is highest in the shallow waters, with a maximum AR of 4000 spec. m−2 yr−1 on the shelf outside the OMZ. The minimum in the benthic foraminifera frequencies found at the lower OMZ boundary is in contrast to a maximum in relative abundance of planktic foraminifera, the latter group making up to 80 % of the sand fraction. We note also an increase in the AR of planktic foraminifera and planktic foraminiferal debris, thus causing the dilution effect on the benthic foraminifera. In contrast to other high-productivity areas, siliceous microfossils (diatoms, radiolarians, silicoflagellates, sponge spicules) are rare in the sand fraction of the surface sediments. In general, the abundance and relative frequencies of siliceous tests in the coarse fraction increase with depth; however no clear pattern is seen, possibly because of the low numbers of less than 100 spec. g−1 . The accumulation of inorganic particles can be interpreted mostly as precipitates of small oxides/oxihydroxides (Law et al., 2009) at the lower OMZ. Pyrite was found sporadically www.biogeosciences.net/11/3107/2014/ inside the spherical tests of the planktic foraminifer Orbulina universa. 4.3.2 High accumulation rates of bulk sediment fractions A general decrease of SRs, and hence ARs, is observed with increasing water depth. Individual stations at the shelf edge show a large variability with the highest AR of bulk sediment of > 4000 mg m−2 d−1 (Fig. 5a). In contrast, the two sites 42 KG and 44 KG immediately below display only half of that flux. MC5 shows a slightly higher AR bulk of 2600 mg m−2 d−1 . On the shelf, ARs are highest and show largest variability. MC6 (543 m, Fig. 1) from the same region is the uppermost core on the slope and displays a relatively low AR bulk of 1110 mg m−2 d−1 (Table 2). The deeper sites (e.g., 39 KG) are situated on the Makran side of the Hab transect. Also, these sites show, in general, a decrease of AR bulk, with a decline with water depth and distance from the shelf from > 2700 to ∼ 1100 mg m−2 d−1 (Fig. 5a). The negative trend is typical for all bulk components (Fig. 5b, c) except for the sediment concentration of OC (Fig. 5d) that is distinctly lowered at the uppermost sites at the Indus side of the investigated Hab transect. In contrast, CaCO3 has very high relative and absolute accumulation rates on the shelf, where its relative fraction is about 25 %. At greater depths, that fraction decreases to less than 15 %. 4.3.3 High-flux events (HFEs) during winter caused incomplete trap time series The longest trap series (EPT2; see Table 2) collected from late spring (May 1995) to late winter, but stopped abruptly in February 1996. An extremely high bulk particle flux of 2000 to > 5000 mg m−2 d−1 was also measured two years before, in early 1994, in shallow traps EPT1 and WPTs (Fig. 6). WPTd (1470 m) of the same period had the shortest collection period of less than three months, between October 1993 and mid-January 1994 (Table 2). WPTd apparently was not directly affected by the extreme flux during winter. Collection stopped, although no increase in flux is noticed for that trap. Biogeosciences, 11, 3107–3120, 2014 3114 H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan Table 2. List of fluxes of lithic matter, calcium carbonate (CaCO3 ), and organic carbon (OC) in the water column (sediment traps EPT1, EPT2, WPTs and WPTd) and reconstructed accumulation rates (ARs) at the seafloor for 16 short cores at water depths between 196 and 1970 m. (a) Water column particle flux and composition Trap Trap depth (m) Sampling start Sampling end Sampling (d) Total flux (mg m−2 d−1 ) Lith. flux (mg m−2 d−1 ) CaCO3 flux (mg m−2 d−1 ) Opal flux (mg m−2 d−1 ) OC flux (mg m−2 d−1 ) Lith. (%) CaCO3 (%) Opal (%) OC (%) EPT1-01 EPT1-02 EPT1-03 EPT1-04 EPT1-05 EPT1-06 590 590 590 590 590 590 15/10/93 06/11/93 28/11/93 20/12/93 11/01/94 02/02/94 05/11/93 27/11/93 19/12/93 10/01/94 01/02/94 23/02/94 22 22 22 22 22 22 504 680 1086 4848 4298 3501 282 464 769 3621 3234 2609 108 138 164 713 639 523 45 43 73 351 297 256 38 19 45 91 71 63 55.96 68.28 70.77 74.68 75.25 74.53 21.5 20.3 15.1 14.7 14.9 14.9 8,9 6.3 6.8 7.2 6.9 7.3 7.56 2.84 4.11 1.87 1.65 1.80 EPT2-01 EPT2-02 EPT2-03 EPT2-04 EPT2-05 EPT2-06 EPT2-07 EPT2-08 EPT2-09 EPT2-10 EPT2-11 EPT2-12 590 590 590 590 590 590 590 590 590 590 590 590 05/05/95 30/05/95 24/06/95 19/07/95 13/08/95 07/09/95 02/10/95 27/10/95 21/11/95 16/12/95 10/01/96 04/02/96 29/05/95 23/06/95 18/07/95 12/08/95 06/09/95 01/10/95 26/10/95 20/11/95 15/12/95 09/01/96 03/02/96 28/02/96 24 24 24 24 24 24 24 24 24 24 24 24 1678 501 688 188 1337 2444 160 142 136 276 4296 4077 1081 326 484 126 1012 1841 106 90 88 190 3270 3124 404 96 109 32 148 225 25 26 24 44 598 530 111 42 51 15 107 226 12 10 9 20 269 281 45 21 24 8 39 84 9 9 8 12 89 79 64.45 65.07 70.43 67.05 75.67 75.34 66.11 63.30 65.15 68.78 76.11 76.63 24.1 19.1 15.8 17.2 11.1 9.2 15.6 18.5 17.7 16.0 13.9 13.0 6.6 8.3 7.5 8.2 8.0 9.3 7.8 7.2 6.6 7.2 6.3 6.9 2.70 4.20 3.50 4.20 2.91 3.44 5.83 6.16 5.90 4.43 2.06 1.93 WPTS1-01 WPTS1-02 WPTS1-03 WPTS1-04 WPTS1-05 WPTS1-06 534 534 534 534 534 534 15/10/93 06/11/93 28/11/93 20/12/93 11/01/94 02/02/94 05/11/93 27/11/93 19/12/93 10/01/94 01/02/94 23/02/94 22 22 22 22 22 22 213 528 413 1424 5105 1564 116 362 230 994 3870 1094 57 97 103 224 738 258 18 33 39 127 334 143 13 21 23 44 90 38 54.32 68.43 55.61 69.79 75.82 69.95 26.5 18.3 25.0 15.7 14.4 16.5 8.4 6.2 9.5 9.0 6.5 9.1 5.94 3.89 5.50 3.09 1.77 2.46 WPTD1-01 WPTD1-02 WPTD1-03 WPTD1-04 1466 1466 1466 1466 15/10/93 06/11/93 28/11/93 20/12/93 05/11/93 27/11/93 19/12/93 10/01/94 22 22 22 22 353 485 471 199 205 332 292 119 74 84 94 45 39 37 46 21 20 18 22 8 58.18 68.48 61.90 59.53 20.9 17.3 19.9 22.5 10.9 7.7 9.7 10.7 5.57 3.62 4.69 4.02 (b) Seafloor sediment accumulation rates and composition. Cruise METEOR 32/2 METEOR 32/2 SONNE 90 SONNE 90 METEOR 32/2 METEOR 32/2 SONNE 90 SONNE 90 SONNE 90 SONNE 90 METEOR 32/2 SONNE 90 SONNE 90 SONNE 90 SONNE 90 SONNE 90 Core Latitude Longitude Water depth (m) Bulk AR (mg m−2 d−1 ) Lith. AR (mg m−2 d−1 ) CaCO3 AR (mg m−2 d−1 ) Opal AR (mg m−2 d−1 ) OC AR (mg m−2 d−1 ) Lith. (%) CaCO3 (%) Opal* (%) OC* (%) MC2 MC3 44 KG 42 KG MC5 MC6 39 KG 53 KG 58 KG 76 KG MC1 143 KG 72 KG 46 KG 144 KG 79 KG 24◦ N 37.90 24◦ N 37.90 24◦ N 37.00 24◦ N 36.50 24◦ N 38.40 24◦ N 39.20 24◦ N 50.00 24◦ N 48.60 24◦ N 46.60 24◦ N 40.60 24◦ N 44.80 24◦ N 45.00 24◦ N 39.10 24◦ N 33.20 24◦ N 36.40 24◦ N 36.00 66◦ E 06.40 66◦ E 04.60 66◦ E 01.50 65◦ E 59.00 66◦ E 02.90 66◦ E 01.00 65◦ E 55.00 65◦ E 55.00 65◦ E 49.20 65◦ E 41.20 65◦ E 46.60 65◦ E 44.30 65◦ E 45.40 65◦ E 41.80 65◦ E 35.10 65◦ E 35.10 196 247 258 313 386 543 704 825 876 1168 1266 1281 1403 1657 1943 1970 4090 4178 2093 2096 2663 1053 2740 2611 2630 912 2047 1156 1370 926 1022 1663 2703 1930 1563 1611 2036 832 2252 2133 2142 751 1678 946 1110 1120 846 1378 1178 910 396 351 454 175 318 328 332 115 250 146 187 117 119 193 167 201 112 101 137 55 159 129 141 45 94 63 73 53 56 96 75 92 48 55 66 28 38 44 40 9 31 12 13 8 11 11 66.08 72.46 74.69 76.85 76.46 78.99 82.20 81.71 81.44 82.27 82.00 81.84 81.03 81.74 82.78 82.89 28.1 20.8 18.0 16.0 16.3 14.3 11.0 12.0 12.0 12.0 12.0 12.0 13.0 12.0 11.0 11.0 4.0 4.6 5.1 4.6 4.9 4.5 5.5 4.7 5.1 4.7 4.5 5.2 5.1 5.4 5.2 5.5 1.79 2.11 2.20 2.53 2.37 2.25 1.31 1.61 1.44 0.99 1.50 0.95 0.88 0.86 0.98 0.60 Water depth (m) Local SR (cm ka−1 ) Inorg. AR (spec. m−2 d−1 ) Megaf. AR (spec. m−2 d−1 ) Silic. AR (spec. m−2 d−1 ) PF-Frag. AR (spec. m−2 d−1 ) P.F. AR (spec. m−2 d−1 ) B.F. AR (spec. m−2 d−1 ) PP (mg m−2 d−1 ) O2 -BW* (mg L−1 ) 196 247 258 313 386 543 704 825 876 1168 1266 1281 1403 1657 1943 1970 205 272 139 144 183 100 167 144 150 56 100 56 67 44 44 78 74 8 4 13 0 0 3 5 3 1 2 0 3 0 3 0 10 470 3033 92 281 152 43 79 42 21 2 16 2 15 2 4 0 4 63 2 4 21 11 25 5 34 4 23 8 4 1 10 12 33 42 21 6 8 18 14 10 37 52 68 7 22 1 7 3 1137 806 347 358 535 281 258 272 650 267 158 73 74 13 38 63 4434 3681 1082 1025 927 815 471 329 1323 38 100 8 27 15 35 47 711 711 709 708 714 716 758 753 739 717 731 730 714 697 706 706 0.08 0.07 0.07 0.08 0.06 0.05 0.07 0.07 0.11 0.19 0.37 0.38 1.36 1.60 1.97 1.99 (b) ff. Core MC2 MC3 44 KG 42 KG MC5 MC6 39 KG 53 KG 58 KG 76 KG MC1 143 KG 72 KG 46 KG 144 KG 79 KG Suthhof et al., 2000. Primary productivity PP ( Antoine et al., 1996). **Bottom water oxygen O2 -BW (von Rad et al., 1995). Biogeosciences, 11, 3107–3120, 2014 www.biogeosciences.net/11/3107/2014/ H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan Inorg. 0 Silic. Megaf. 20 0 20 40 60 0 P.F. P.F.debris 20 0 20 3115 0 20 40 B.F. 60 80 20 40 60 0 500 1000 OMZ 1500 Water depth (m) 2000 2500 A) % 3000 1 10 100 1000 1 100 10000 0 10 100 1 10 100 1000 10 1000 10 100 10000 0 500 OMZ 1000 1500 2000 2500 B) Spec. g-1 3000 0 40 80 0 6000 12000 0 50 100 0 50 0 500 1000 1500 0 2000 4000 0 500 OMZ 1000 1500 2000 C) AR spec. m-2 d -1 Figure 4. Main components of the coarse fraction (> 150 µm) along the Hab transect (see Fig. 1b, c). Inorg. stands for lithogenic and authigenic particles (quartz, concretions, pyrite, etc.), Megaf. for megafossils (mussels, snails, fish, etc.), Silic. for siliceous biogenic particles (radiolarians, diatoms, sponge spicules), P.F. debris for fragments of planktic foraminifera shells, P.F. for intact planktic foraminifera, and B.F. for benthic foraminifera and fragments. The rows display (A) relative abundance (%), (B) concentration (number of spec. g−1 ), and (C) accumulation rates (AR) of spec. m−2 d−1 of the six categories. OMZ stands for oxygen minimum zone. Here we define the onset of the winter high-flux period due to the dramatic shift in bulk flux as high-flux events (HFEs). According to the definition of Schott et al. (2009), that period in the northern Arabian Sea temporally coincides with the late winter NE monsoon period, although the timing of the start of the HFE is slightly different for the two winters sampled. In EPT2 the HFE shift is observed from collection period 10 to 11 (10 January 1996). In EPT1 an increase is noticed in sampling cup 3, and the strong shift is from cup 4 to cup 5 (20 December 1993). For the deployment of the same year further offshore (WPTs and WPTd were programmed to sample flux simultaneously to EPT1 closer onshore, Table 2), the start of an HFE is fully recorded at the shallow trap WPTs, but occurs one collection period (22 days) later (cup 4). The vertical trap particle flux in the Hab area is about 10 times higher when compared to the open ocean areas of the Arabian Sea (Haake et al., 1993). Moreover, the flux rates and composition within the water column (Fig. 6b), which could be determined only for a limited period of time, agree rather well in dimensions, with the accumulation rates and fractions determined at the seafloor (Fig. 5). Both the water column and seafloor estimates of the bulk flux amount to several thousand mg m−2 d−1 , and the CaCO3 and OC fluxes range from several tens to several hundreds of mg m−2 d−1 (Table 2). An elevated trap flux during and after summer (SW) monsoon (in July/August according to the definition of Schott et al., 2009) could be recorded in EPT2. It is characterized by a higher concentration of OC (∼ 2%), whereas all winter fluxes have www.biogeosciences.net/11/3107/2014/ relatively low OC (∼ 1%). An analysis of the winter coarse fraction of EPT2 hints at the origin of the HFE matter: small benthic foraminifera, presumably remobilized from the shelf and upper slope, constitute up to 10 % of the foraminiferal flux during the winter season (Schulz et al., 2002). 5 Discussion Based on our detailed studies of the sediment profiles, the finding of more than one layer leaves some uncertainty on the exact patterns displayed by the sedimentation rates. We described different colors and thicknesses of layers, and observe presumably different event layers deposited one on top of the other at one site (58 KG). This refutes exact synchronism of that marker bed. We suggest that the red and gray layers are almost synchronous. These are separated by 5–10 thin sediment laminae (Staubwasser and Sirocko, 2001), since they were deposited by a similar turbidite or turbid-layertype gravitational deposition process around AD 1905 ± 10. We believe that our correlative stratigraphic approach by two event layers may be superior to reconstructions based on individually dated records. Especially those cores from below the OMZ would be at risk of being biased by bioturbation effects. Thick turbidites clearly form the major sediment fraction on the central Makran and clearly contrast with the thin seasonal sediment laminae that characterize the Pakistan Hab (and Indus) margin sections (Bourget et al., 2010; von Rad et al., 1995, their Ormara transect). To this end, the few thick layers in the shallow sediment sections of the Hab area, Biogeosciences, 11, 3107–3120, 2014 3116 H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan a) 6000 Fall intermonsoon FLUX (OC)/Flux (Lith.) 5000 Winter monsoon HFE 4000 EPT2 EPT1 3000 2000 WPTs 1000 ? WPTd 0 260 280 300 320 340 360 380 400 420 440 Days (start January 1,1993; traps EPT1, WPTs, WPTd) Days (start January 1,1995; trap EPT2) b) sandwiched between laminated sediments, are unique and offer a good opportunity for correlation. We find a rather low, stable accumulation of organic carbon below the OMZ with an OC AR of only ∼ 10 mg m−2 d−1 . At these sites, we also notice the very low but uniform accumulation of benthic foraminifera. The parallel accumulation rates may document the close relationship between the benthic faunas and available OC (food). This may be valid only for the aerobic fauna. However, approaching the lower margin of the OMZ, significant changes in the faunal composition have been noticed (Schumacher et al., 2007). Zobel (1973) observed a strong reduction of benthic foraminifera at the lower boundary and explained the gap in the study’s percentage data (Fig. 4) by a benthic community change between a more aerobic deeper water Bulimina aculeata facies and an OMZ Buliminacea biofacies. The high density of benthic foraminifera of ∼ 200– 1000 spec. m−2 yr−1 (live + dead fauna) in the upper part of the OMZ is in line with the more recent observations showing that benthic foraminifera, as part of the meiofauna, play a central role in OM cycling. In particular, the form Uvigerina ex. gr. semiornata is present in the upper part of the Pakistan OMZ (Larkin and Gooday, 2009). However, off the Indus margin to the south of Karachi, these authors observe high benthic foraminiferal densities at the deep OMZ, whereas acBiogeosciences, 11, 3107–3120, 2014 Fall intermonsoon Winter monsoon EPT1 WPTs FLUX (OC)/Flux (Lith.) Figure 5. Estimated accumulation rates (AR) of selected bulk parameters vs. water depth. Note that upper stations are on the Indus side, whereas the deeper cores are from the Makran margin. (A) AR bulk, (B) AR CaCO3 , (C) AR organic carbon OC and (D) sedimentary organic carbon OC (%). OMZ stands for oxygen minimum zone. 0.15 0.10 WPTd EPT2 ? 0.05 HFE 0.00 260 280 300 320 340 360 380 400 420 440 Days (start January 1,1993; traps EPT1, WPTs, WPTd) Days (start January 1,1995; trap EPT2) Figure 6. Summarized details of flux recording prior and during the winter high-flux events (HFEs) from the four sediment traps (EPT2, EPT1, WPTs and WPTd). Note that the latter three have synchronous collection periods (see Table 2). (A) Differences in the timing of increasing and peak flux in the shallow traps EPT and WPTs). (B) Composition of settling flux (exemplified by the ratio of OC / lithic). All traps display similar compositions prior to and during the HFEs, culminating in an increase of lithic matter. The shaded area marks the transition from fall intermonsoon (shaded) to winter monsoon (dark gray). The horizontal axis marks days after 1993 for EPT1, WPTs and WPTd, and days after 1995 for EPT2. www.biogeosciences.net/11/3107/2014/ H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan cumulation rates in the Hab transect on the eastern Makran show a pattern of decline with water depth, as depicted by most other sediment properties. Within the OMZ, sediment profiles are largely intact as they lack the smoothing effect of bioturbation. As expected, sedimentation rates are highly variable in an active continental margin setting. It should be mentioned here that neither the OC AR nor the concentration data of our data set can provide us with an estimate of the key parameter, which is organic carbon burial efficiency, to assess any potential preservational effect on OC by the OMZ. Here we provide estimates on the age and porosity (which parallels density) of the sediments, as well as on the O2 content of the local bottom waters (Table 2), but we miss a measure for diffusion of O2 into the sediment to comment on whether there are differences in O2 exposure time. Hartnett et al. (1998) indicated that oxygen exposure time is important, especially in explaining the decline in OC below the OMZ. However, at no site, even with the sediment trap data, can we determine the percentage of OC delivered to the sediments that is actually buried. As a main finding, our profiles show that the upper boundary of the OMZ is characterized by high OC fluxes at the outer shelf and downslope transport of allochthonous matter. OC AR (mg m−2 d−1 ) and OC (%) strongly decline from the upper slope ∼ 400 m to ∼ 800–900 m, and again towards the lower margin of the OMZ, where both parameters reach low values at 1200–1300 m and do not decrease further below. Down to 2000 m water depth, OC ARs are ∼ 10 mg m−2 d−1 (∼ 1.5 % of the regional primary productivity estimate at the sea surface (720 ± 17 mg OC m−2 d−1 )). On the outer shelf, at 250 m water depth, that fraction is ∼ 10 % (Table 2). We suggest that the local shelf drainage system of the Hab area may be unique, dominated by a seasonally variable deposition of extremely fine-grained matter downslope. Only very little coarse materials like macrofossil shells or clastic material escape from the shelf (Fig. 4). Our suggestion is in line with the findings to the south of our region, where organic matter on the seafloor of the Indus area is found strongly degraded in the laminated sediments (Woulds and Cowie, 2009; Jeffreys et al., 2009; Vandewiele et al., 2009). However, there is little information on the various sources and pathways of matter. By the comparatively weak bottom currents and the very low ventilation, and by of the local drainage system, the Hab sedimentary environment contrasts with the other highly productive, low-oxygen margin zones of the Arabian Sea basin to the west and to the southeast. For example, OC, C / N, hydrogen index data and mineralogical indicators do not match to the mid-depth oxygenminimum patterns along the highly productive, steep Oman margin (Pedersen et al. 1992). Attendant downslope reworking and sediment winnowing were found to best explain the OC patterns found there, but sediment laminae today are absent from the Oman margin. As reported previously by Calvert et al. (1995), laminae are also absent, and there is mawww.biogeosciences.net/11/3107/2014/ 3117 jor variability in OC content and sediment grain size at any given depth within the OMZ on the Indian margin. However, compared to the eastern Makran, on both margins there is sufficient oxygen to permit some level of bioturbation, even at the OMZ core, and/or insufficient seasonal change in inputs, resulting in the lack of laminae there. The key point that can be inferred from the two studies of Pedersen et al. (1992) and Calvert et al. (1995) is the large range of grain size and OC contents, associated with strong bottom currents on the upper slope and shelf, ultimately resulting in more sandy sediments on the West Indian and Oman margins. Coarse sediments and ripple beds were also clearly observed at the OMZ core off India (von Stackelberg, 1972; Petra Heinz, personal communication, 2014). On the Indus margin, the OC maximum is found at the lower boundary of the OMZ (Cowie et al. 1999), as in many transects off India and Oman. Therefore in these settings, local near-bottom transport and changes in sediment texture are obviously the dominant control on OC distribution, overriding any oxygen effect there. In contrast, our data exhibit an OC maximum near the upper boundary of the OMZ. The preservation of laminae (Fig. 2) and the completeness of the sediment profiles (Fig. 3) point to a rather stable sedimentary environment and to the reduced importance of near-bottom sediment transport off the Makran coastal zone (Fig. 4). Strong and event-like near-bottom resedimentation events (except the marked clay-silt turbidite) were not recorded from the sediments during the past ∼ 100 years in the Hab area and on the upper Indus slope. However, thin silty event deposits of light-gray color are eventually interbedded between the seasonal laminae couplets (see “C layers” in Schulz et al. (1996) and von Rad et al. (2002b); core photos provided as a supplement). Unique sedimentary conditions may prevail not only across the slope (Fig. 2–5) but also in the water column off the Hab transect. We emphasize that the extremely finegrained, organic and clastic material resuspended from shallow depths during the winter season is one of the main sedimentary factors that causes the OC maximum near the upper continental margin, and plays an important role for the formation of the Pakistan laminated sediments. Von Rad et al. (2002b) report that there is continuous lamination in the Holocene core 56KA at station 39 KG. For at least the past ∼ 5000 years, more than 50 wt% (up to 65 %) of the bulk matter flux was in the clay fraction, with a small median of only 1.8 µm. The phenomenon of a high water column flux on the shelf and on the upper margin can also be expected in other margin environments, but has rarely been monitored because of the small number of sediment trap moorings installed at coastal and continental margin sites to record the highly dynamic flux. A good overview of sediment traps issues can be taken from the global trap compilation study of Žaric et al. (2006). Only 1 out of 41 casts was found from a shallow open margin setting with water depths < 1000 m. Biogeosciences, 11, 3107–3120, 2014 3118 H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan For the Pakistan margin, we assume that, due to winter HFE, sediment trap cups and possibly even the funnels were filled with sediment. In both sediment trap time series EPT1 and WPTs of 1993/1994, only the first six cups were filled (Andruleit et al., 2000; Schulz et al., 2002) because trap functions terminated. By the same suspension events traps and finally became clogged (Fig. 6). Whatever the exact reason was for the termination of the records, the three traps (except WPTd) collected a huge amount of suspended matter immediately before they stopped functioning (Fig. 6a). The exact timing of increases in flux is different for the three traps, but it is possible that fluxes followed a temporal and spatial evolution: an HFE in 1993/1994 at EPT1 started in the last weeks of 1993 and was recorded in trap WPTs offshore only in early 1994. We speculate that the HFE of that winter evolved in the coastal and/or shelf margin zones and subsequently spread away from the coast to record the high fluxes 20 km further offshore at WPTs one sampling period later. A very similar winter flux pattern was observed two years later in EPT2. The increase in fluxes recorded in 1995/1996 started relatively late compared to EPT1. The average flux composition for the winter period was very similar between the more coastal sites and WPTs trap further offshore (Fig. 6b), and the winter flux material strongly resembles that of the sediments (Table 2). We speculate that the upper water column in the late winter season must have been loaded to more than 30 km distance from the shelf edge (2004 m seafloor depth) with a rather uniform sediment suspension. Based on our bulk sediment AR below the traps, we stress that a “normal” flux of sinking matter (as is observed in EPT2 before the HFE) would not be high enough to explain the deposition of several 1000 mg m−2 d−1 of laminated sediment offshore. As an example, we speculate that the longest time series, EPT2, must have received an HFE up to 20 000 to 30 000 mg m−2 d−1 . This large winter flux is needed to balance the low fluxes of only ∼ 750 mg m−2 d−1 recorded during spring, summer and fall in order to reach the 2000 to 2500 mg m−2 d−1 of flux that we reconstructed from sediment stations MC1 and 58 KG below the EPT moorings. However, because of the lack of direct observations, we can only speculate on the temporal and spatial dimensions of the winter resuspension process. 6 Conclusions 1. A quantitative reconstruction of sediment fluxes using a combination of high-quality short sediment cores with sediment trap results leads to a better understanding of the roles of low-oxygen conditions and of the horizontal vs. vertical particle transport across the easternmost part of the upper Makran and the northern section of the Indus margin. As a major finding of the study, we present here precise estimates of the mass accumulation rates and a detailed characterization of the different sediment fractions. We estimate that the total error for the sediment flux reconstructions is < 20 %. 2. Profiling of 210 Pb, sediment AR determinations and 14 C dating has previously been done on the Pakistan, Indus and other margins, though not with the spatial coverage and precision of AR estimates presented in our study. Our studies indicate that the trends in organic carbon concentration seen on the Hab/Makran transects are not typical of other Arabian Sea margins. The identification of an upper-margin organic carbon maximum at ∼ 400 m is supported by sediment flux estimates spanning the entire OMZ down to 2000 m. These estimates indicate that the main source of the material is from shallow water depths, from the outer shelf and uppermost continental slope, even though the texture of bulk sediments on the slope appears to be extremely finegrained (clay-sized). 3. From a source-to-sink perspective, our investigations imply that important processes of sediment resuspension and dispersal must take place within the water column. Water column processes dominate over the factors of near-bottom transport and preservation. In the outer shelf area the bulk sediment accumulation rates are > 4000 mg m−2 d−1 , more than twice the rates at 1000 m water depth. These high rates compare best with those from the sediment traps EPT and WPT during the winter season deployed at only ∼ 600 m trap depth. 4. The high-flux events (HFE) documented in our traps suggest that large-scale sediment suspensions of > 5000 mg m−2 d−1 spread offshore at the upper slope in the (NE) monsoon season. This suggests a strong control by the winter monsoon processes on the sedimentary regime and on the formation of the laminated sediments on the Pakistan margin in the northeastern Arabian Sea. The Supplement related to this article is available online at doi:10.5194/bg-11-3107-2014-supplement. Biogeosciences, 11, 3107–3120, 2014 www.biogeosciences.net/11/3107/2014/ H. Schulz and U. von Rad: Vertical and lateral flux on the continental slope off Pakistan Acknowledgements. The authors thank the editor, Greg Cowie, for helpful comments and the two anonymous reviewers for their thorough and constructive reviews. We thank Birgit Stenschke and Martina Schmidtke for laboratory assistance, Helmut Kawohl and Rainer Goergens for obtaining high-quality sediment cores, and Captain Papenhagen and Captain Kull and their skilled crews for help and good collaboration onboard R.V. SONNE and R.V. METEOR. The first author wishes to express his sincere thanks to the Hamburg group of marine biogeochemistry – Venu Ittekkot, Andreas Suthhof, Tim Jennerjahn, Jörg Tiemann, Martin Wiesner and Birgit Gaye – for discussions and support. 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https://openalex.org/W2797834202	https://zenodo.org/records/2426175/files/article.pdf	English	null	78. Easter Island: Script.	Man	1,904	public-domain	1,853	Wiley, Royal Anthropological Institute of Great Britain and Ireland are collaborating with JSTOR to digitize, preserve and extend access to Man Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms 78. Easter Island: Script. Author(s): O. M. Dalton Source: Man, Vol. 4 (1904), pp. 115-116 Published by: Royal Anthropological Institute of Great Britain and Ireland Stable URL: http://www.jstor.org/stable/2839700 Accessed: 27-06-2016 04:09 UTC East ty 0. M. Dalton, M.A., F.S.A. Easter Island: Script. B ar 10 Easter Island: Script. Bty 0. M. Dalton, M.A., F.S.A. The short article on an inscribed tablet published in MAN for the present year 10 (1904.1), though it contained a certain number of references,madeno pretence of providing a bibliography for the script of Easter island. Mr. N. W. Thomas has suggested that it might be worth while to add a supplementary series of references, and this I am now enabled to do, thanks to his co-operation. The follo-wing list, arranged chronologically, has been selected from a larger one, a number of references having been omitted as containing information at second or third hand, or mere allusions to the script without scientific value. The literature relating to Rapa Nui and its antiquities is, of course, very extensive, but many writers ignore the tablets altogether or just note their existence, so that the books and papers with which a student of the " hieroglyphs " has to deal are not so numerous as might have been expected. I must express my regret for the omission of all mention in the previous article of the two tablets at Vienna described in Dr. Max Haberlandt's communication in the Vienna Mittheilungena noted below. The papers in which the script is treated at any length, or which contain suggestive matter, are marked in the list with an asterisk. It is not to be hoped that this is an exhaustive bibliography, but it may prove of some little use to students of the ethnography of the Pacific:- Easter Island Script. Bty 0. M. Dalton, M.A., F.S.A. The short article on an inscribed tablet published in MAN for the present year 10 (1904.1), though it contained a certain number of references,madeno pretence of providing a bibliography for the script of Easter island. Mr. N. W. Thomas has suggested that it might be worth while to add a supplementary series of references, and this I am now enabled to do, thanks to his co-operation. The follo-wing list, arranged chronologically, has been selected from a larger one, a number of references having been omitted as containing information at second or third hand, or mere allusions to the script without scientific value. 78. Easter Island: Script. Author(s): O. M. Dalton Source: Man, Vol. 4 (1904), pp. 115-116 Published by: Royal Anthropological Institute of Great Britain and Ireland Stable URL: http://www.jstor.org/stable/2839700 Accessed: 27-06-2016 04:09 UTC 78. Easter Island: Script. Author(s): O. M. Dalton Source: Man, Vol. 4 (1904), pp. 115-116 Published by: Royal Anthropological Institute of Great Britain and Ireland Stable URL: http://www.jstor.org/stable/2839700 Accessed: 27-06-2016 04:09 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Wiley, Royal Anthropological Institute of Great Britain and Ireland are collaborating with JSTOR to digitize, preserve and extend access to Man Wiley, Royal Anthropological Institute of Great Britain and Ireland are collaborating with JSTOR to digitize, preserve and extend access to Man 7-78. 19 N. it proves that a complete refoundation was made; the stones below the sand-bed having been left sunk in the ground and ignored, while a layer of sand was laid over them for founding a new temple. Thus the view, which we exposed in the digging, of many courses of stones separated by three or four beds of sand can be read off as recording the founading of so many separate temples. g Though no whole dynasty of kings has been brought to light, as in our work at Abydos, yet the fresh and strong evidence about the early date of some rulers and styles, and the recovery of two of the finest monuments knowni, and the plans of the series of temples on a great site, make this year another landmark in the clearing of Egyptian history. Beside the temple site we worked also in the town, entirely on burnt houses of Roman age. Thuis we have been able to date a long series of terra-cotta figures, which are of much finer work than was expected in the third and fourth centuries A.D. And a tolerablv complete corpus of Romano-Egyptian lamps was made, and the degradation of types traced throughout more than 1,000 varieties. This may, perhaps, be more fully described here in future. W. M. FLINDERS PETRIE. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:09:20 UTC All use subject to http://about.jstor.org/terms No MA 04. 1872. Revue Maritime, Vol. 35, pp. 105 if., L'1le de P6ques. (Short official report by Admiral de Lapelin to the Minister of Marine, mentioning visit of the Flore of the French Pacific squadron to Easter Island in 1872. This is followed by a translation of the report of Captain Gana of the Chilian vessel O'Higgins. The translator's notes on p. 121 relate to the tablets and give facts in support of their comparatively recent origin.) g g 1873. Iswestia (proceedings of the Russian Geographical Society, Vol. VIII., p. 42). (Article by Miklucho Maclay.) p y y 1873. Compte-rendt de l'Acade'mie des Inscriptions et Belles-lettres, pp. 151-155. (A. de Longperier discusses casts of inscriptions in the collection of the Bishop of Axieri.) g 1876. Zeitschrift fiur Ethnologie, Vol. VIII., p. 37 ff. (Philippi, Uber die Hiero- glyphen der Osterinsel, ec.: Mentions the receipt at tile Santiago Museum of a third example of the script, in addition to the original two tablets, casts of which had been sent to Europe.) 1878. Le Tour du Monde. (Article by Pinart, who reproduces one of the Santiago casts ; the same writer in Bulletin de la Societe de Geographie, Vol. XVI. (1878), p. 203, notices figures carved in relief upon stone buildings.) p g p g 1881. A. B. Meyer, Die Bilderschriften des Ostindischen Archipels und der Siudsee (publication of the Royal Ethnographical Museum at Dresden, printed at Leipzig), p. 7, plate vi. (The inscription of a tablet is reproduced, and the plates giving examples of picture-writing from Pelew, Celebes, &c., are interesting for purposes of contrast and comparison.) 1881. Globus, Vol. XL., p. 375. (Notice by Andree of the preceding work by Meyer.) 1883. A. Bastian, Zur Naturwissenschaftlichen Behandlungsweise de) Psychologie, Berlin, pp. 212 ff., with plate reproducing the example in the Museum ftir Volkerkunde, Berlin. 1883.* Ymer (Stockholm), Vol. III., pp. 182 ff. (H. Stolpe, part of his article on Pdsk-6n, or Easter Island. This interestinig paper is unfortunately written in Swedish, and is therefore accessible to a comparatively small number of students. It may be mentioned that the same author has treated the tattooing of the Easter Islanders in the Abhandlungen of the Dresden Zoological and Anthropological Museum, Festschrift, 1899, No. 6.) 1886.* Mittheilungen der Anthropologischen Gesellschaft in Wien, pp. 97ff., plate x. (M. Haberlandt, ber Schrifttafeln von der Osterinsel. Discusses the relation of the tablets to genealogy. East The literature relating to Rapa Nui and its antiquities is, of course, very extensive, but many writers ignore the tablets altogether or just note their existence, so that the books and papers with which a student of the " hieroglyphs " has to deal are not so numerous as might have been expected. I must express my regret for the omission of all mention in the previous article of the two tablets at Vienna described in Dr. Max Haberlandt's communication in the Vienna Mittheilungena noted below. The papers in which the script is treated at any length, or which contain suggestive matter, are marked in the list with an asterisk. It is not to be hoped that this is an exhaustive bibliography, but it may prove of some little use to students of the ethnography of the Pacific:- 1866. Globus (published at Brunswick), Vol. XIII., p. 313. (Short account of Eyraud's nine months" stay in the island; contains the statement that there were tablets in almost every house.) 1870. Ibid., Vol. XVII., p. 248. (Palmer's account of visit of H.M.S. Topaze.) 1870. Zeitschrift der Gesellschaft fur Erdkunade, Berlin, p. 469. (Letter of Dr. Philippi to Professor Bastian mentioning the tablets, and the report of Captain Gana, commander of the O'Higgins, the Chilian vessel which visited the island in 1870.) 1871.* Ibid., pp. 548-551. (Meinicke, Die Holztafein von Rapanui. The writer protests against any theory of American influence, and rejects the view that the script was due to a now extinct race. He believes the inscriptions to represent geneatlogies.) g g 1872.* Ibid., Vol. VII., pp. 78-81. (Miklucho Maclay, Uber die Rohaurogo, oder die Holztafeln von Rapanui. Draws attention to the similarity of characters on the tablets to designs carved on stone statues, &c. The article is followed by remarks by Professor Bastian (pp. 81-9), who suggests that the subjects of the inscriptions are not merely genealogies, and may be ceremonial recitations for use at particular feasts.) [ 115 ] This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:09:20 UTC All use subject to http://about.jstor.org/terms No They are probably not of high antiquity, and are more important from the point of view of psychology than history.) p p p y gy y 1889. The American Naturalist, Vol. XXIII., p. 882. (W. Hough mentions th tablets in an article on the visit of the Mohician.) 1893. Bulletin de Giographie historique et descriptive (Comite' des travaux, historiques et scientifques, Annee 1892), Paris, 1893, pp. 240-270. (Bishop Jaussen's posthumous memoir, L'Ile de Paques, edited by the R.-P. Ildefonse Alazard and containing the vocabularies. See MAN, 1904, p. 2.) 1895.* Le Museon, Revue Internationale, published at Louvain, Vol. XIV., p. 415 ff., and Vol. XV., pp. 68-73. (C. de Harlez, reproduction of inscription from a tablet with the translation given by Metoro to Bishop Jaussen. De Harlez declares it to be une suite d'images indetpendantes l'une de l'autre, and not a continuous narrative at all. Cf. MAN, 1904, p. 4.) 1899. Comptes-rendus de la Socie'te de Getographie, Paris, pp. 169-176. (Captain H. Vere Barclay's, R.N., account of visit of H.M.S. Topaze. On p. 175 Captain Barclay says that the characters on the tablets are not the same as those on the statues, &c. He believes in a connection with S. America and suspects Maya influence.) 0. M. DALTON. [ 116 1 [ 116 1 This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:09:20 UTC All use subject to http://about.jstor.org/terms
https://openalex.org/W2938375103	https://iris.unive.it/bitstream/10278/3749827/1/epjconf_up2019_05012.pdf	English	null	Real-time observation of the intravalley spin-flip process in single-layer WS<sub>2</sub>	EPJ web of conferences	2,019	cc-by	1,951	Real-time observation of the intravalley spin-flip process in single-layer WS2 In our experiment, spin-polarized carriers are photo-injected by a circularly polarized pump resonant to the A exciton transition, while their temporal evolution is detected by a co- circularly polarized probe pulse resonant with either the A or B excitons (red and blue curves in the central panel in Fig. 1). When the probe is resonant with the A exciton, we observe an instantaneous (i.e. limited by the temporal resolution ~ 100fs) build-up of the differential transmission (ΔT/T) signal. This positive signal (photo-bleaching, PB) arise from Pauli-blocking in the A excitonic state On the other hand when the probe is resonant with the B exciton the intravalley spin- In two-dimensional Transition Metal Dichalcogenides (TMDs) interactions with light are dominated by strongly bounded excitons due to the low-dimensional quantum confinement[1]. In Mo and W based TMDs, the large spin-orbit coupling lifts the spin degeneracy of the valence (VB) and the conduction bands (CB) giving rise to the A and B interband excitonic transitions[2]. Theory predicts the CB spin-ordering in Mo-based TMDs to be different from W-based TMDs [3]. In the former case, the spins of the electrons in the lowest CB and in the highest VB at K/K’ are parallel, giving rise to an optically bright A exciton state while in the latter case they are antiparallel, resulting in a dark A exciton at a lower energy than the bright one (intravalley dark exciton, see left panel in Fig.1). The presence of dark excitons has been revealed indirectly from the observation of anomalous quenching of the PL emission at low temperature in single-layer (1L)-WSe2[4]. Here we use two-colour helicity-resolved pump-probe spectroscopy to directly resolve the intravalley spin-flip process of the photoexcited electrons in the CB of 1L-WS2. In our experiment, spin-polarized carriers are photo-injected by a circularly polarized pump resonant to the A exciton transition, while their temporal evolution is detected by a co- circularly polarized probe pulse resonant with either the A or B excitons (red and blue curves in the central panel in Fig. 1). When the probe is resonant with the A exciton, we observe an instantaneous (i.e. limited by the temporal resolution ~ 100fs) build-up of the differential transmission (ΔT/T) signal. This positive signal (photo-bleaching, PB) arise from Pauli-blocking in the A excitonic state. © 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/). , (201 E Web of Conferences PJ 9) 205 0 UP 2018 5012 , (201 E Web of Conferences PJ 9) 205 0 UP 2018 5012 https://doi.org/10.1051/e onf /201920 pjc 505012 Real-time observation of the intravalley spin-flip process in single-layer WS2 Zilong Wang1, Alejandro Molina-Sanchez2, Patrick Altmann1, Davide Sangalli3, Domenico De Fazio4, Giancarlo Soavi4, Ugo Sassi4, Federico Bottegoni1, Franco Ciccacci1, Marco Finazzi1, Ludger Wirtz5, Andrea Ferrari4, Andrea Marini3, Giulio Cerullo1,6 and Stefano Dal Conte1,* 1Dipartimento di Fisica, Politecnico di Milano, Piazza L. da Vinci 32, I-20133 Milano, Italy 2Institute of Materials Science (ICMUV), University of Valencia, Catedràtico Beltràn 2, E-46980, Valencia, Spain 1Dipartimento di Fisica, Politecnico di Milano, Piazza L. da Vinci 32, I-20133 Milano, Italy 2Institute of Materials Science (ICMUV), University of Valencia, Catedràtico Beltràn 2, E-46980, Valencia, Spain 3Istituto di Struttura della Materia (ISM), CNR, Via Salaria Km 29.3, I-00016 Monterotondo Stazione, Italy 3Istituto di Struttura della Materia (ISM), CNR, Via Salaria Km 29.3, I-00016 Monterotondo Stazione, Italy 4Cambridge Graphene Centre, University of Cambridge, 9 JJ Thomson Avenue, Cambridge CB3 0FA, United Kingdom 4Cambridge Graphene Centre, University of Cambridge, 9 JJ Thomson Avenue, Cambridge CB3 0FA, United Kingdom g, , 6IFN-CNR, Piazza L. da Vinci 32, I-20133 Milano, Italy Abstract. We use helicity-resolved transient absorption spectroscopy to track intravalley scattering dynamics in monolayer WS2. We find that spin- polarized carriers scatter from upper to lower conduction band by reversing their spin orientation on a sub-ps timescale. In two-dimensional Transition Metal Dichalcogenides (TMDs) interactions with light are dominated by strongly bounded excitons due to the low-dimensional quantum confinement[1]. In Mo and W based TMDs, the large spin-orbit coupling lifts the spin degeneracy of the valence (VB) and the conduction bands (CB) giving rise to the A and B interband excitonic transitions[2]. Theory predicts the CB spin-ordering in Mo-based TMDs to be different from W-based TMDs [3]. In the former case, the spins of the electrons in the lowest CB and in the highest VB at K/K’ are parallel, giving rise to an optically bright A exciton state while in the latter case they are antiparallel, resulting in a dark A exciton at a lower energy than the bright one (intravalley dark exciton, see left panel in Fig.1). The presence of dark excitons has been revealed indirectly from the observation of anomalous quenching of the PL emission at low temperature in single-layer (1L)-WSe2[4]. Here we use two-colour helicity-resolved pump-probe spectroscopy to directly resolve the intravalley spin-flip process of the photoexcited electrons in the CB of 1L-WS2. Real-time observation of the intravalley spin-flip process in single-layer WS2 On the other hand, when the probe is resonant with the B exciton, the intravalley spin- flip scattering process (grey arrow in left panel in Fig. 1) from upper to lower CB states causes a delayed formation (on a time scale τrise) of the PB signal (blue curve in Fig.1b). We find that at 77 K this process occurs on a sub-ps time scale (i.e. ~ 200 fs). , (201 E Web of Conferences PJ 9) 205 0 UP 2018 5012 https://doi.org/10.1051/e onf /201920 pjc 505012 Fig. 1. (Left panel) Sketch of the band structure of 1L-WS2 around the K point of the Brillouin zone. The pump and probe pulses have the same circular polarization. The pump is resonant with the A exciton while the probe is resonant with either the A (degenerate configuration) or B exciton (non- degenerate configuration). Only the non-degenerate configuration is sketched for sake of clarity. Only excitons in K point are excited because of the valley dependent optical selection rules for circular polarization. The intravalley scattering process (grey arrow) from upper to lower CB state causes the reduction of the absorption of the probe beam by Pauli blocking and results in a delayed formation of the bleaching signal around the B exciton. (Central panel) The red and blue curves are the T/T signal measured in 1L-WS2 at 77K around the A and B excitonic transitions, following excitation of the A transition. For degenerate pump and probe energies, the build-up dynamics is pulse-width limited, while the non-degenerate configuration displays a delayed formation. In the inset, T/T around the B exciton peak is multiplied by 4 to highlight the finite build up time. The grey line is the cross correlation function between the pump and probe pulses. (Right panel) Temperature dependence of the B exciton rise time rise. Fig. 1. (Left panel) Sketch of the band structure of 1L-WS2 around the K point of the Brillouin zone. The pump and probe pulses have the same circular polarization. The pump is resonant with the A exciton while the probe is resonant with either the A (degenerate configuration) or B exciton (non- degenerate configuration). Only the non-degenerate configuration is sketched for sake of clarity. Only excitons in K point are excited because of the valley dependent optical selection rules for circular polarization. Real-time observation of the intravalley spin-flip process in single-layer WS2 2 2 , (201 E Web of Conferences PJ 9) 205 0 UP 2018 5012 https://doi.org/10.1051/e onf /201920 pjc 505012 Fig. 2. (Left panel) Calculated band structure of 1L-WS2. The spin-orbit splitting of the valence band in the K point is in good agreement with the experimental value. In the inset the calculated splitting of the CB (i.e. 26 meV) is reported on an enlarged scale. (Right panel) Temporal evolution of the number of carriers in the upper and lower CB state (respectively K,↑ and K,↓) around the K point after excitation by a 100 fs pump pulse (green curve) centered around the A exciton. Fig. 2. (Left panel) Calculated band structure of 1L-WS2. The spin-orbit splitting of the valence band in the K point is in good agreement with the experimental value. In the inset the calculated splitting of the CB (i.e. 26 meV) is reported on an enlarged scale. (Right panel) Temporal evolution of the number of carriers in the upper and lower CB state (respectively K,↑ and K,↓) around the K point after excitation by a 100 fs pump pulse (green curve) centered around the A exciton. Real-time observation of the intravalley spin-flip process in single-layer WS2 The intravalley scattering process (grey arrow) from upper to lower CB state causes the reduction of the absorption of the probe beam by Pauli blocking and results in a delayed formation of the bleaching signal around the B exciton. (Central panel) The red and blue curves are the T/T signal measured in 1L-WS2 at 77K around the A and B excitonic transitions, following excitation of the A transition. For degenerate pump and probe energies, the build-up dynamics is pulse-width limited, while the non-degenerate configuration displays a delayed formation. In the inset, T/T around the B exciton peak is multiplied by 4 to highlight the finite build up time. The grey line is the cross correlation function between the pump and probe pulses. (Right panel) Temperature dependence of the B exciton rise time rise. Moreover, the strong dependence of τrise on the lattice temperature strongly suggests that this relaxation process is mediated by phonons (right panel of Fig. 1). In order to get more insight into the origin and the dynamics of the measured intravalley relaxation process, we use first-principles calculations based on non-equilibrium many- body perturbation theory [5]. y p y The band structure and the static absorption spectrum of 1L-WS2 are first calculated using density functional theory within local density approximation by taking into account the strong spin-orbit interaction (left panel in Fig. 2). The temporal dynamics is obtained by solving the Kadanoff–Baym equation for the one–body density matrix. In order to simulate our pump-probe experiments, we impose that the photoexcited carriers occupy only the upper CB state of the K valley at time zero, while at later times they can scatter into other electronic states by emitting phonons. y g p The right panel in Fig. 2 reports the temporal dynamics of the occupations of upper and lower CB states. This shows that the upper CB states are quickly depleted by efficient scattering processes mediated by phonons, while the lower ones are progressively filled on a temporal scale close to our experimental value. In conclusion, our results shed light on the intravalley spin relaxation process in 1L-WS2, determining the formation of intravalley dark excitons on a sub-ps timescale. Dark excitons determine the light emission efficiency of 1L-TMDs. For this reason, understanding the formation process of dark excitons is of valuable importance for designing 1L-TMDs based optoelectronic devices such light emitting diodes and polarized photon emitters. References 1. G. Wang, A. Chernikov, M. M. Glazov, T. F. Heinz, X. Marie, T. Amand, and B. Urbaszek, Rev. Mod. Phys. 90, 021001 (2018) 2. T. Cheiwchanchamnangij and W. R. L. Lambrecht, Phys. Rev. B 85, 205302 (2012) 3. K. Kosmider, J. W. Gonzalez and J. Fernandez-Rossier, Phys. Rev. B 88, 245436 (2013). 3. K. Kosmider, J. W. Gonzalez and J. Fernandez-Rossier, Phys. Rev. B 88, 245436 (2013). 4. X.-X. Zhang, Y. You, S. Y. F. Zhao and T. F. Heinz, Phys. Rev. Lett. 115, 257403 (2015). 4. X.-X. Zhang, Y. You, S. Y. F. Zhao and T. F. Heinz, Phys. Rev. Lett. 115, 257403 (2015). 5. A. Molina-Sanchez, D. Sangalli, L. Wirtz and A. Marini, Nano Lett. 17, 4549 (2017) 3 3
https://openalex.org/W4393667607	https://jwd.unram.ac.id/index.php/jwd/article/download/174/153	Indonesian	null	PENTINGNYA PENGUASAAN TENSES BAHASA INGGRIS DALAM BERBICARA	Jurnal Warta Desa	2,022	cc-by-sa	3,094	PENTINGNYA PENGUASAAN TENSES BAHASA INGGRIS DALAM BERBICARA I Gede Nika Wirawan1* 1Program Studi Sistem Informasi, ITB STIKOM Bali *Coresponding-Author : nika_wirawan@stikom-bali.ac.id ABSTRAK. Pengabdian kepada mesayarakat ini dilaksanakan pada kelompok belajar desa Sangeh Badung dalam bentuk kegiatan pengajaran percakapan bahasa Inggris. Penggunaan tenses dalam melakukan percakapan Bahasa Inggris sangat penting. Sehingga tujuan kegiatan pengabdian kepada masyarakat ini adalah untuk meningkatkan keterampilan berbicara Bahasa Inggris siswa-siswa di kelompok belajar desa Sangeh-Badung. Metode pengajaran yang diterapkan dalam pelatihan tersebut yaitu metode Communicative Language Taeching in Secondary Level. Pelatihan Bahasa Inggris ini merupakan Kerjasama antara kursus Konsultan Bahasa dan Prodi Bahasa Inggris. Di samping itu, kegiatan ini dilaksanakan secara offline guna mendapatkan hasil kegiatan pembelajaran yang maksimal. Pada kegiatan pengabdian tersebut, siswa kelompok belajar di desa Sangeh sangat antusias dan aktif untuk mengikuti pembelajaran atau pelatihan percakapan Bahasa Inggris. Siswa pada kelompok belajar ini diberikan beberapa pemahaman tentang tenses Bahasa Inggris yang sering dipakai, yaitu simple present tense, simple future tense dan simple past tense. Selain itu juga peserta pengabmas ini juga melakukan latihan percakapan langsungd dengan pengajar sehingga pengajar bisa memantau langsung terhadap proses perkembangan peserta didiknya. ______________________ Kata Kunci: Percakapan bahasa Inggris, pengajaran, Kelompok Belajar ______________________ Kata Kunci: Percakapan bahasa Inggris, pengajaran, Kelompok Belajar ______________________ Kata Kunci: Percakapan bahasa Inggris, pengajaran, Kelompok Belajar ABSTRACT. This community service was carried out in Sangeh Badung village study group in the form of English conversation teaching activities. The use of tenses in conducting English conversation is very important. Therefore, this community service activity aimed to improve the English-speaking skills of students in the Sangeh-Badung village study group. The teaching method applied in the training was the Communicative Language Taeching in Secondary Level method. This English training was a collaboration between the Language Consultant course and the English Study Program. In addition, this activity was carried out offline in order to get the maximum results of learning activities. In these service activities, study group students in Sangeh village were very enthusiastic and active in participating in English conversation learning or training. Students in this study group were given some understanding of the English tenses that were often used, namely simple present tense, simple future tense and simple past tense. In addition, these community service participants also conducted direct conversation exercises with teachers. Therefore, teachers could directly monitor the development process of their students. ______________________ Keywords: English Conversation, Teaching, Study Group Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 PENDAHULUAN Bahasa Inggris merupakan media yang sangat penting dalam komunikasi di Indonesia. Bahasa Inggris berperan sebagai bahasa global atau dunia. Menurut Crystal (2003), peran bahasa Inggris dapat dilihat dalam berbagai bidang seperti pada dunia pariwisata, pendidikan, ekonomi, maupun dalam komunikasi sehari-hari. Ini menandakan bahwa penguasaan terhadap bahasa Inggris di Indonesia sangat penting sekali. Bahasa Inggris memeiliki peranan yang sangat penting dan dapat digunakan dalam tujuan tertentu seperti tujuan akademis, peningkatan karir, dan juga untuk kebutuhan perjanan ke luar negeri. Bahasa Inggris merupakan bahasa asing pertama yang 21 p-ISSN 2776-2572; e-ISSN 2685-2101 Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 bisa dipelajari sebagai mata pelajaran wajib dari sekolah menengah pertama hingga perguruan tinggi di Indonesia (Nurcahyani, 2020). Menteri Pendidikan dan Kebudayaan memasukkan mata pelajaran bahasa Inggris mulai dari sekolah dasar karena bahasa Inggris memiliki peranan yang sangat penting di Indonesia. Pembelajaran Bahasa Inggris terhadap para siswa untuk mengenal dasar-dasar bahasa Inggris dimulai sejak dini. Menurut Kusuma, Zakaria & Djuwita (2017), dalam berbicara mengenai bahasa Inggris, terdapat 4 (empat) keterampilan yang harus dikuasai dalam belajar bahasa Inggris. Terdapat empat keterampilan yang diperlukan dalam penguasaan Bahasa Inggris tersebut yaitu keterampilan mendengarkan, keterampilan berbicara, keterampilan membaca, dan keterampilan menulis. Keterampilan mendengarkan dan membaca disebut receptive skills karena pelajar hanya menerima dan memahami materi pelajaran yang diajarkan. Sedangkan keterampilan berbicara dan menulis disebut dengan istilah productive skills karena siswa diharapkan mampu memproduksi bahasa yang telah dikuasai. Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 bisa dipelajari sebagai mata pelajaran wajib dari sekolah menengah pertama hingga perguruan tinggi di Indonesia (Nurcahyani, 2020). Menteri Pendidikan dan Kebudayaan memasukkan mata pelajaran bahasa Inggris mulai dari sekolah dasar karena bahasa Inggris memiliki peranan yang sangat penting di Indonesia. Pembelajaran Bahasa Inggris terhadap para siswa untuk mengenal dasar-dasar bahasa Inggris dimulai sejak dini. Menurut Kusuma, Zakaria & Djuwita (2017), dalam berbicara mengenai bahasa Inggris, terdapat 4 (empat) keterampilan yang harus dikuasai dalam belajar bahasa Inggris. Terdapat empat keterampilan yang diperlukan dalam penguasaan Bahasa Inggris tersebut yaitu keterampilan mendengarkan, keterampilan berbicara, keterampilan membaca, dan keterampilan menulis. PENDAHULUAN Keterampilan mendengarkan dan membaca disebut receptive skills karena pelajar hanya menerima dan memahami materi pelajaran yang diajarkan. Sedangkan keterampilan berbicara dan menulis disebut dengan istilah productive skills karena siswa diharapkan mampu memproduksi bahasa yang telah dikuasai. Keterampilan yang paling penting untuk dikuasai adalah keterampilan berbicara merupakan keterampilan yang paling penting untuk dikuasai dalam dunia komunikasi sehari-hari. Menurut Brown (2000), mengajar keterampilan berbicara bukanlah merupakan hal mudah yang dipengaruhi oleh sejumlah faktor. Ada lima hambatan yang ditemuai oleh seorang pelajar dalam mengikuti proses pembelajaran di antaranya (1) siswa masih merasa takut untuk membuat kesalahan, (2) siswa nampaknya tidak memperoleh kesempatan untuk terlibat dalam aktivitas berbicara dalam kelas, (3) terdapat siswa yang pasif, sehingga siswa merasa enggan untuk terlibat dalam aktivitas speaking, (4) topik pembelajaran yang disajikan kurang menarik bagi siswa, (5) siswa kadang- kadang masih berbicara kecil dan pelan sehingga sulit didengar oleh guru. Seorang pengajar harus bersifat sabar ketika sedang mendampingi peserta pengabmas untuk mendmpingi kegiatan percakapan Bahasa Inggris agar mendapatkan hasil yang maksimal. Pengajar jugaperlo mendorong sisiwa agar bisa berbicara lancar dalam Bahasa Inggris dan siswa merasa nyaman ketika didampingi oleh pengajar dalam melakukan praktik percapakan Bahasa Inggris tersebut. Tarigan (2013) mengungkapkan bahwa berbicara adalah kemampuan untuk mengucapkan bunyi-bunyi artikulasi atau kata-kata untuk mengekspresikan, menyatakan serta menyampaikan pikiran, gagasan, dan perasaan. Lebih lanjut, Budiarso (2019) mengungkapkan bahwa, terdapat tiga kegiatan yang harus diterapkan oleh seorang pengajar dalam aktivitas dalam pelajaran keterampilan. Siswa sebaiknya mampu membuat kalimat sendiri dalam Bahasa Inggris mereka bisa meningkatkan kemampuan Bahasa Inggrisnya. Metode asking and answering merupakan metode pembelajaran yang dapat dilakukan ole siswa secara bergiliran. Peserta pelatihan percakapan Bahasa Inggris ini nampak sangat antusias dalam proses belajar mengajar yang dilaksanakan dengan bertujuan untuk meningkatkan kemampuan percakapan Bahasa Inggris dasar peserta. SOLUSI PERMASALAHAN Dalam memberikan pelatihan percakapan bahasa Inggris ini, pengajar menggunakan metode asking and answering, group work, telling stories. Metode yang diterapkan dalam pelatihan percakapan Bahasa Inggris ini yaitu Communicative Language Taeching in Secondary Level. Communicative Language Teaching merupakan metode yang didasarkan pada target mengajar dan pembelajaran bahasa untuk komunikasi kehidupan nyata dan efektif (Seraj & Mamun, 2011). Metode yang digunakan dalam proses pelatihan percakapan Bahasa Inggris dasar ini yaitu metode persuasive. Metode yang dilakukan yaitu metode pengumpulan data observasi berdasarkan dengan mengamati fenomena yang terjadi di tempat pelatihan Bahasa Inggris. Pelatihan Bahasa Inggris dasar merupakan bentuk pelatihan yang diberikan kepada peserta pengabmas ini. Peserta pelatihan bahasa Inggris ini nampak sangat antusias di dalam mengikuti pelatihan. Metode pendekatan kualitatif juga digunakan dalam laporan Pengabdian kepada Masyarakat tentang percakapan Bahasa Inggris dasar ini. Bogdan dan Taylor dalam Moleong (2010:4) mengungkapkan bahwa 22 p-ISSN 2776-2572; e-ISSN 2685-2101 Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 metodologi penelitian kualitatif sebagai prosedur penelitian yang menghasilkan data deskriptif berupa kata-kata tertulis atau lisan dari orang-orang dan perilaku yang bisa kita amati. Teori yang dikemukakan oleh Muhammad (2011: 30) mengungkapkan bahwa penelitian kualitatif merupakan jenis penelitian dengan paradigma pos-positivism, yang memiliki tujuan untuk menafsirkan objek yang akan diteliti dengan menggunakan beberapa metode dan dilaksanakan pada latar alamiah. Ismawati (2012: 9) lebih lanjut mengungkapkan bahwa, penelitian kualitatif merupakan metode kualitatif karena beberapa faktor. Pengajar dalam melakukan pelatihan percakapan Bahasa Inggris dasar ini juga menggunakan gerakan tubuh untuk mempercepat pemahaman peserta didik dalam melakukan pelatihan Bahasa Inggris ini. Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 metodologi penelitian kualitatif sebagai prosedur penelitian yang menghasilkan data deskriptif berupa kata-kata tertulis atau lisan dari orang-orang dan perilaku yang bisa kita amati. Teori yang dikemukakan oleh Muhammad (2011: 30) mengungkapkan bahwa penelitian kualitatif merupakan jenis penelitian dengan paradigma pos-positivism, yang memiliki tujuan untuk menafsirkan objek yang akan diteliti dengan menggunakan beberapa metode dan dilaksanakan pada latar alamiah. Ismawati (2012: 9) lebih lanjut mengungkapkan bahwa, penelitian kualitatif merupakan metode kualitatif karena beberapa faktor. Pengajar dalam melakukan pelatihan percakapan Bahasa Inggris dasar ini juga menggunakan gerakan tubuh untuk mempercepat pemahaman peserta didik dalam melakukan pelatihan Bahasa Inggris ini. HASIL DAN PEMBAHASAN Para peserta pengabmas ini diberikan pemahaman tentang beberapa tenses Bahasa Inggris supaya para peserta pengabmas memiliki pemahaman yang baik tentang beberapa tenses yang digunakan untuk melakukan percakapan Bahasa Inggris dasar ini. Beberapa tenses yang digunakan untuk melakukan percakapan antara lain simple present, simple future tense dan simple past tense yang bisa digunakan untuk melakukan percakapan dengan peserta pengabmas. Para peserta pengabmas mempraktikkan percakapan bahasa Inggris dasar ini secara langsung suppaya bisa meningkatkan kemampuan berbicara Bahasa Inggris dasar ini. Adapun penggunaan tenses yang digunakan yaitu simple present tense karena tenses ini dianggap tepat digunakan bagi pemula yang ingin belajar Bahasa Inggris. Berikut ini merupakan contoh kalimat dalam present tense yang diberikan sebagai pembekalan kepada para peserta pengabmas tersebut. Tenses dalam bentuk present tenses ini digunakan untuk meningkatkan kemahiran dalam penguasaan tenses sebelum melakukan percakapan dalam Bahasa Inggris. Berikut ini merupakan contoh klausa dalam simple present tense yang diberikan kepada peserta pengabmas berikut ini. • I get up at 7:00 every day. • Then I take a shower, get dressed and eat breakfast. • I leave my house at 8:00 and ride the bus to school. • I start class at 9:00 on Monday, and 10:00 on Thursday. • I usually have lunch at 1:00. • I finish class at 3:30 p.m. • On Saturday, I sleep in until 11:00. Berikut ini merupakan contoh beberapa pertanyaan yang digunakan dalam simple present tense. Berikut ini merupakan contoh beberapa pertanyaan yang digunakan dalam simple present tense. • What time do you usually wake up? • What do you do between classes? • What time do you finish work? Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License • What do you do in the evening? • What do you do in the evening? Para peserta pengabmas sebelum melakukan percakapan Bahasa Inggris dengan temannya, mereka terlebih dahulu akan dilatih dengan membuat kalimat Bahasa Inggris dengan menggunakan simple present tense dan juga didampingi oleh pengajar. Pendampingan dalam proses pembelajaran percakapan bahasa Inggris ini untuk memantau langsung tentang kemampuan Bahasa Inggris para peserta pengabmas secara langsung. Setelah diberikan pembekalan tentang 23 p-ISSN 2776-2572; e-ISSN 2685-2101 Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 pemahaman simple present tense ini, peserta pengabmas ini akan melatih percakapan dengan menggunakan simple present tense berikut ini. Dengan mengasah percakapan ini para peserta pengabmas akan terbiasa melakukan percakapan dengan beberapa pertanyaan yang digunakan tersebut. Para peserta pengabmas ini nampak sangat menikmati kegiatan percakapan yang diberikan oleh pengajar percakapan Bahasa Inggris dasar ini. Proses pelatihan Bahasa Inggris dasar ini dimulai dengan melakukan greeting dalam bahasa Inggris, kemudian dilanjutkan dengan mempelajari kosa-kata yang berhubungan dengan kegiatan sehari-hari. Peserta pengabmas juga kemudian melakukan percakapan bahasa Inggris keperawatan. Kemudian dilanjutkan dengan mengajari peserta pengabmas tentang bagaimana mengucapkan kosa kata yang diberikan. Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 pemahaman simple present tense ini, peserta pengabmas ini akan melatih percakapan dengan menggunakan simple present tense berikut ini. Dengan mengasah percakapan ini para peserta pengabmas akan terbiasa melakukan percakapan dengan beberapa pertanyaan yang digunakan tersebut. Para peserta pengabmas ini nampak sangat menikmati kegiatan percakapan yang diberikan oleh pengajar percakapan Bahasa Inggris dasar ini. Proses pelatihan Bahasa Inggris dasar ini dimulai dengan melakukan greeting dalam bahasa Inggris, kemudian dilanjutkan dengan mempelajari kosa-kata yang berhubungan dengan kegiatan sehari-hari. Peserta pengabmas juga kemudian melakukan percakapan bahasa Inggris keperawatan. Kemudian dilanjutkan dengan mengajari peserta pengabmas tentang bagaimana mengucapkan kosa kata yang diberikan. pemahaman simple present tense ini, peserta pengabmas ini akan melatih percakapan dengan menggunakan simple present tense berikut ini. Dengan mengasah percakapan ini para peserta pengabmas akan terbiasa melakukan percakapan dengan beberapa pertanyaan yang digunakan tersebut. Para peserta pengabmas ini nampak sangat menikmati kegiatan percakapan yang diberikan oleh pengajar percakapan Bahasa Inggris dasar ini. Proses pelatihan Bahasa Inggris dasar ini dimulai dengan melakukan greeting dalam bahasa Inggris, kemudian dilanjutkan dengan mempelajari kosa-kata yang berhubungan dengan kegiatan sehari-hari. • What do you do in the evening? Peserta pengabmas juga kemudian melakukan percakapan bahasa Inggris keperawatan. Kemudian dilanjutkan dengan mengajari peserta pengabmas tentang bagaimana mengucapkan kosa kata yang diberikan. Peserta pengabmas juga diberikan pembekalan tentang pemahaman simple past tense dalam bahasa Inggris. Sebelum melakukan percakapan, peserta pengabmas diberikan pembekalan tentang pemahaman tenses ini guna mematangkan teori sebelum melakukan percakapan dengan menggunakan simple past tense. Nampak para peserta pengabmas melakukan percakapan bahasa Inggris dengan menggunakan simple past tense. Berikut ini merupakan contoh paragrap dengan menggunakan simple past tense yang digunakan oleh peserta pengabmas sebelum melakukan percakapan Bahasa Inggris dasar. Last week I went to Bali Zoo Park by public transportation. I left home at 8 o’clock in the morning and arrived there around 9 AM. I spent some hours there. I saw many kinds of animals. There were many wilds animals and many visitors too. It was nice since the weather was not bad. Gambar 1. Terlihat peserta pengabmas sedang mempelajari naskah percakapan. Gambar 1. Terlihat peserta pengabmas sedang mempelajari naskah percakapan. Pada Gambar 1 tersebut terlihat para peserta pengabmas sedang melakukan pemahaman simple present tense sebelum melakukan percakapan Bahasa Inggris dasar ini. Nampak peserta yang antusias ketika melakukan percakapan Bahasa Inggris dasar ini. Mereka didampingi oleh pengajar ketika melakukan percakapan Bahasa Inggris dasar ini. 24 p-ISSN 2776-2572; e-ISSN 2685-2101 Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 Gambar 2. Terlihat peserta pengabmas sedang mempraktikkan percakapan Bahasa Inggris dasar dengan menggunakan simple past tense. Gambar 2. Terlihat peserta pengabmas sedang mempraktikkan percakapan Bahasa Inggris dasar dengan menggunakan simple past tense. Gambar 2. Terlihat peserta pengabmas sedang mempraktikkan percakapan Bahasa Inggris dasar denga menggunakan simple past tense. Gambar 2. Terlihat peserta pengabmas sedang mempraktikkan percakapan Bahasa Inggris dasar denga menggunakan simple past tense. Sebelum melakukan percakapan dengan menggunakan simple past tense, para peserta pengabmas ini diberikan pemahaman teori tentang simple past tense agar peserta pengabmas ini bisa melakukan percakapan Bahasa Inggris dasar dengan lancar. Beberapa pertanyaan dalam simple past tense juga diberikan kepada peserta pengabmas ini guna melatih kemampuan berbicara peserta. KESIMPULAN Para peserta pengabmas ini diberikan pelatihan percakapan bahasa Inggris dasar dengan menggunakan simple present tense, simple past tense sehingga bisa meningkatkan kemampuan berbicara dalam Bahasa Inggrisnya. Proses pelatihan Bahasa Inggris dasar ini dimulai dengan memberikan greeting dalam bahasa Inggris, kemudian dilanjutkan dengan mempelajari kosa-kata yang berhubungan dengan Bahasa Inggris tingkat dasar. Peserta pengabmas juga kemudian melakukan percakapan bahasa Inggris keperawatan. Kemudian dilanjutkan dengan mengajari peserta pengabmas tentang bagaimana mengucapkan kosa kata yang diberikan. Sebelum melakukan percakapan Bahasa Inggris dasar ini, peserta pengabmas diberikan pembekalan tentang pemahaman tenses ini guna mematangkan teori sebelum melakukan percakapan dengan menggunakan simple past tense. Nampak para peserta pengabmas melakukan percakapan bahasa Inggris dengan menggunakan simple past tense. Pendampingan dalam proses pembelajaran percakapan bahasa Inggris ini dilakukan untuk memantau langsung tentang kemampuan Bahasa Inggris para peserta pengabmas secara langsung. Setelah diberikan pembekalan tentang pemahaman simple present tense ini, peserta pengabmas ini akan melatih percakapan dengan menggunakan simple present tense. Creative Commons Attribution-ShareAlike 4.0 International License ( ) p g g g g ( ) g Budiarso, I. (2019). Analisis Metode Communicative Language Teaching Terhadap Kemampuan Berbicara Bahasa Inggris Guru-Guru SMK dan SMP Islam Mandiri Bojong Gede Bogor. Jurnal SAP, 3(3), 239–247. Jurnal Warta Desa (JWD) is licensed under a REFERENSI Brown, H. D. (2000). Principles of language learning and teaching (4th ed.). Longman. Budiarso, I. (2019). Analisis Metode Communicative Language Teaching Terhadap Kemampuan Berbicara Bahasa Inggris Guru-Guru SMK dan SMP Islam Mandiri Bojong Gede Bogor. Jurnal SAP, 3(3), 239–247. Crystal, D. (2003). English as a Global Language (2nd ed.). Cambridge University Press. Encylopedic Dictionary of Applied Linguistics. Blackwell. Kusuma, D., Zakaria, & Djuwita, P. (2017). Manajemen Pembelajaran Bahasa Inggris dalam 25 25 p-ISSN 2776-2572; e-ISSN 2685-2101 Jurnal Warta Desa (JWD) is licensed under a Jurnal Warta Desa (JWD) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 Vol. 4, No. 1, April 2022, pp. 21~26 DOI: 10.29303/jwd.v4i1.174 Meningkatkan Kemampuan Writing Siswa SMP. Manajer Pendidikan, 11(3), 254–262. Nurcahyani, A. (2020). Peningkatan Keterampilan Berbicara Bahasa Inggris Melalui Media Flash Card Siswa Kelas 3 SDN Putat 02, Geger, Madiun Tahun Ajaran 2019/2020. Institut Agama Islam Negeri Ponorogo. Richards, J. C., & Rodgers, T. S. (2001). Approaches and Methods in Language Teaching (2nd ed.). Cambridge University Press. Richards, J. C., & Rodgers, T. S. (2001). Approaches and Methods in Language Teaching (2nd ed.). Cambridge University Press. Seraj, P. M. I., & Mamun, M. A. A. (2011). 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https://openalex.org/W4244346783	https://aprp.msal.ru/jour/article/download/381/351	Russian	null	On the Issue of Legislative Regulation of Transferability of Objects of Civil Rights in the Context of the RF Civil Code Reform	Aktualʹnye problemy rossijskogo prava	2,017	cc-by	3,928	© Борисова Л. В., 2017 * Борисова Лилия Владимировна, кандидат юридических наук, доцент, Финансовый университет при Правительстве Российской Федерации, доцент Департамента «Правовое регулирование экономиче- ской деятельности» grpfa@yandex.ru 125993, Россия, г. Москва, Ленинградский просп., д. 49 ГРАЖДАНСКОЕ И СЕМЕЙНОЕ ПРАВО ГРАЖДАНСКОЕ И СЕМЕЙНОЕ ПРАВО Л. В. Борисова* О проблеме законодательного регулирования оборотоспособности объектов гражданских прав в контексте реформы Гражданского кодекса Российской Федерации Аннотация. В статье выделены и проанализированы отдельные проблемы законодатель- ного регулирования оборотоспособности объектов гражданских прав. Обращено внимание на недостатки законодательной техники, допущенные при формулировании ст. 129 ГК РФ, ее неполноту и противоречие с соответствующими правилами специального законода- тельства. В частности, отмечено различие содержания термина «оборотоспособность» в данной норме и иных законодательных актах, показана необходимость расширения крите- рия ограничения оборотоспособности объектов гражданских прав в совершении сделок по специальному разрешению. Автором предложены возможные варианты разрешения данных проблем и противоречий. Высказаны критические замечания относительно исключения из содержания ст. 129 ГК РФ правил об обращении изъятых из оборота объектов, которые в силу Федерального закона от 2 июля 2013 г. № 142-ФЗ «О внесении изменений в подраздел 3 раздела I части первой Гражданского кодекса Российской Федерации» признаны ограниченны- ми в обороте. В связи с этим предложено законодательное выделение необоротоспособной группы объектов, которые, по мнению автора, могут разграничиваться на три категории: необоротоспособные объекты в силу присущих им естественных свойств, указаний закона, либо неразрывной связи с определенным участником гражданского оборота. Статья может быть полезной научным работникам, преподавателям, аспирантам, ма- гистрантам, студентам юридических факультетов, а также всем интересующимся про- блемами гражданского права. Ключевые слова: оборотоспособность, объект гражданских прав, вещи, изъятые из оборота объекты, ограниченные в обороте объекты, гражданский оборот, правовой режим, сделки, специальное законодательство, правопреемство. Актуальные проблемы российского права. 2017. № 1 (74) январь DOI 10.17803/1994-1471.2017.74.1.126-131 Согласно ч. 1 ст. 8 Конституции Российской Федерации в Российской Федерации гаранти- руются единство экономического простран- ства, свободное перемещение товаров, услуг и финансовых средств, поддержка конкуренции, свобода экономической деятельности. вых средств распространяется и на действия, опосредующие оборот объектов гражданских прав. Пункт 1 ст. 129 Гражданского кодекса Российской Федерации (далее — ГК РФ) закре- пляет общее правило их оборотоспособности, согласно которому объекты гражданских прав могут свободно отчуждаться или переходить от одного лица к другому в порядке универ- Конституционное положение о свобод- ном перемещении товаров, услуг и финансо- Актуальные проблемы российского права. 2017. № 1 (74) январь 126 Л. В. Борисова О проблеме законодательного регулирования оборотоспособности объектов гражданских прав Л. В. Борисова Л. В. Борисова О проблеме законодательного регулирования оборотоспособности объектов гражданских прав Не сводится только к отчуждению и охва- тывает все формы использования оборот ору- жия и основных частей огнестрельного оружия (производство, торговля, продажа, передача, приобретение, коллекционирование, экспони- рование, учет, хранение, ношение, перевозка, транспортирование, использование, изъятие, уничтожение, ввоз оружия в Российскую Феде- рацию и вывоз его из Российской Федерации) (ст. 1 Федерального закона от 13.12.1996 № 150-ФЗ «Об оружии»). сального правопреемства (наследование, ре- организация юридического лица) либо иным способом, если они не ограничены в обороте. Из приведенной нормы следует, что, во-первых, понятие оборотоспособности ха- рактеризует отношения динамики, поскольку представляет собой возможность объектов отчуждаться или переходить от одного лица к другому. Такое определение рассматриваемо- го термина соответствует его филологическому значению, где составляющий термин «обо- рот» определяется как движение, круговорот, скорость, движение туда и обратно и т.д. 1, а также сложившемуся в предшествующие годы пониманию оборотоспособности в цивилисти- ческой науке. Например, Е. А. Суханов опре- деляет оборотоспособность как «способность служить объектом имущественного оборота (различных сделок) и менять своих владельцев (собственников)»2. О. Н. Садиков как «допусти- мость совершения сделок и иных действий, направленных на их передачу в рамках граж- данско-правовых отношений»3. По мнению В. А. Белова, оборотоспособность есть призна- ние объектов гражданских прав «товарами — объектами экономического оборота», как их «социальное свойство — меновую стоимость»4 и др. Статья 1 Федерального закона от 22 ноября 1995 г. № 171-ФЗ «О государственном регу- лировании производства и оборота этилово- го спирта, алкогольной и спиртосодержащей продукции» называет оборотом закупку (в том числе импорт), поставку (в том числе экспорт), хранение и розничную продажу. Аналогичная норма содержится в Феде- ральном законе от 2 января 2000 г. № 29-ФЗ «О качестве и безопасности пищевых продук- тов». 4 Белов В.А. Гражданское право: Общая часть: Учебник. М., 2011. Т. II. Лица, блага, факты. С. 278. Так же. см.: Харитонова Ю. С. Комм. к ст. 1180 ГК РФ. Комментарий к Гражданскому кодексу Российской Федера- ции. В 2 т. Т. 1. Части первая, вторая ГК РФ /Под ред. Т.Е. Абовой, А.Ю. Кабалкина. — Ин-т государства и права РАН. Издательство «Юрайт», 2011. А б й 2017 №1 (74) 1 См.: Ожегов С.И., Шведова Н.Ю. Толковый словарь русского языка. М., 1949-1992 ; Бизнес. Толковый словарь. /Общая редакция И.М. Осадчая. М.,1998. /[Электронный ресурс]: Доступ из справ.- правовой си- стемы «Консультант плюс» 2 Гражданское право: Учебник: В 4 т. Общая часть / Под ред. Е. А. Суханова. М.: Волтерс Клувер, 2008. Т. 1. С. 401 3 Комментарий к Гражданскому кодексу Российской Федерации (части первой) (постатейный) /под ред. О.Н. Садикова. М., 2003. С. 315 4 Белов В.А. Гражданское право: Общая часть: Учебник. М., 2011. Т. II. Лица, блага, факты. С. 278. Так же. см.: Харитонова Ю. С. Комм. к ст. 1180 ГК РФ. Комментарий к Гражданскому кодексу Российской Федера- ции. В 2 т. Т. 1. Части первая, вторая ГК РФ /Под ред. Т.Е. Абовой, А.Ю. Кабалкина. — Ин-т государства и права РАН. Издательство «Юрайт», 2011. 1 См.: Ожегов С.И., Шведова Н.Ю. Толковый словарь русского языка. М., 1949-1992 ; Бизнес. Толковый словарь. /Общая редакция И.М. Осадчая. М.,1998. /[Электронный ресурс]: Доступ из справ.- правовой си- стемы «Консультант плюс» 1 См.: Ожегов С.И., Шведова Н.Ю. Толковый словарь русского языка. М., 1949-1992 ; Бизнес. Толковый словарь. /Общая редакция И.М. Осадчая. М.,1998. /[Электронный ресурс]: Доступ из справ.- правовой си- стемы «Консультант плюс» 2 Гражданское право: Учебник: В 4 т. Общая часть / Под ред. Е. А. Суханова. М.: Волтерс Клувер, 2008. Т. 1. С. 401 3 Комментарий к Гражданскому кодексу Российской Федерации (части первой) (постатейный) /под ред. О.Н. Садикова. М., 2003. С. 315 4 Белов В.А. Гражданское право: Общая часть: Учебник. М., 2011. Т. II. Лица, блага, факты. С. 278. Так же. см.: Харитонова Ю. С. Комм. к ст. 1180 ГК РФ. Комментарий к Гражданскому кодексу Российской Федера- Актуальные проблемы российского права. 2017. № 1 (74) январь DOI 10.17803/1994-1471.2017.74.1.126-131 Оборотом признаются купля-продажа (в том числе экспорт и импорт) и иные способы передачи пищевых продуктов, материалов и изделий, их хранение и перевозка. В Федеральном законе от 08.01.98 № 3-ФЗ «О наркотических средствах и психотропных веществах» (ст. 2) под оборотом понимается «культивирование растений; разработка, про- изводство, изготовление, переработка, хране- ние, перевозка, пересылка, отпуск, реализа- ция, распределение, приобретение, использо- вание, ввоз на таможенную территорию Рос- сийской Федерации, вывоз с таможенной тер- ритории Российской Федерации, уничтожение наркотических средств, психотропных веществ и их прекурсоров, разрешенные и контролиру- Во-вторых, определенный в ст. 129 ГК РФ объем понятия «оборотоспособность» ограни- чивается совершением сделок по отчуждению объекта, что не согласуется со специальным за- конодательством. Например, земельные участ- ки, указанные в ст. 27 Земельного кодекса Рос- сийской Федерации (далее — ЗК РФ) не могут быть объектами любых сделок, предусмотрен- ных гражданским законодательством. Актуальные проблемы российского права. 2017. № 1 (74) январь 127 ГРАЖДАНСКОЕ И СЕМЕЙНОЕ ПРАВО емые в соответствии с законодательством Рос- сийской Федерации». рации»); химическое оружие, объекты по хра- нению и уничтожению химического оружия, которые согласно ч. 1 ст. 5 Федерального за- кона от 2 мая 1997 г. № 76-ФЗ «Об уничтоже- нии химического оружия» находятся в исклю- чительном ве́дении Российской Федерации и в федеральной собственности, управление которой осуществляется Правительством Рос- сийской Федерации в установленном поряд- ке; суда с ядерными энергетическими уста- новками (п. 2 ст. 12 КТМ РФ) и др. Учитывая изложенное, считаем, что в опре- делении оборотоспособности объектов граж- данских прав (п. 1 ст. 129 ГК РФ) необходимо использовать более обобщенное и соответ- ствующее специальному законодательству по- нятие «сделки». В связи с принятием Федерального закона от 2 июля 2013 г. № 142-ФЗ «О внесении изме- нений в подраздел 3 раздела I части первой Гражданского кодекса Российской Федерации» исследовательский интерес вызывает пункт 2 ст. 129 ГК РФ, определяющий критерии ограни- чения оборотоспособности объектов граждан- ских прав. Для устранения данного противоречия, считаем, что на законодательном уровне было бы логичным обозначить существо- вание необоротоспособной группы объек- тов, тем более что принадлежность таковых только определенным участникам оборота, выходит за рамки общего филологического и цивилистического понимания термина «обо- ротоспособность», которое, как отмечалось, связывается с отношениями динамики. То есть необоротоспособными следует признать объек- ты, которые могут принадлежать лишь опреде- ленным участникам оборота и не могут быть предметом сделок либо переходить от одних лиц к другим в порядке правопреемства или иным способом. Актуальные проблемы российского права. 2017. № 1 (74) январь DOI 10.17803/1994-1471.2017.74.1.126-131 На наш взгляд, с точки зрения ГК РФ и специального законодательства, воз- можны три категории таких объектов: Анализ специального законодательства в данном отношении позволяет выделить опре- деленные проблемы, обусловленные недо- статками законодательной техники, допущен- ными при формулировании этого пункта. Первое противоречие выявляется уже при оценке норм об обращении изъятых из оборо- та объектов, которые в силу названного феде- рального закона признаны ограниченными в обороте. Во-первых, как следует из специального за- конодательства, ограниченные и до недавнего времени изъятые из оборота объекты, имеют разный правовой режим в части совершения сделок и перехода от одного лица к друго- му. Особенность режима изъятых из оборота объектов заключается в том, что они не могут быть предметом сделок и переходить от одно- го лица к другому в порядке правопреемства либо иным способом, тогда как ограниченные в обороте объекты, к которым таковые теперь отнесены, могут менять своего хозяина при наличии специального разрешения (лицен- зия, квота, свидетельство о государственной регистрации и др.). Например, не могут быть объектами сделок, предусмотренных граж- данским законодательством, названные в п. 4 ст. 27 ЗК РФ земельные объекты; некоторые объекты культурного наследия (п. 10, 12-14 ст. 9 Федерального закона от 25 июня 2002 г. № 73-ФЗ «Об объектах культурного наследия (памятниках истории и культуры) народов Российской Феде- − необоротоспособные объекты в силу естественных свойств, например, неотчужда- емые и непередаваемые иным способом результаты интеллектуальной деятельности и приравненные к ним средств индивидуализа- ции, а также нематериальные блага и личные неимущественные права, принадлежащие гражданину от рождения или в силу закона. В числе естественных свойств, исключающих оборотоспособность данных объектов, мож- но указать на их нематериальный характер, неповторяемость, не возможность измерения с помощью вещно-правовых категорий (вес, масса) и др. ; − необоротоспособные объекты в силу естественных свойств, например, неотчужда- емые и непередаваемые иным способом результаты интеллектуальной деятельности и приравненные к ним средств индивидуализа- ции, а также нематериальные блага и личные неимущественные права, принадлежащие гражданину от рождения или в силу закона. В числе естественных свойств, исключающих оборотоспособность данных объектов, мож- но указать на их нематериальный характер, неповторяемость, не возможность измерения с помощью вещно-правовых категорий (вес, масса) и др. ; − необоротоспособные объекты в силу естественных свойств, например, неотчужда- емые и непередаваемые иным способом результаты интеллектуальной деятельности и приравненные к ним средств индивидуализа- ции, а также нематериальные блага и личные неимущественные права, принадлежащие гражданину от рождения или в силу закона. DOI 10.17803/1994-1471.2017.74.1.126-131 В числе естественных свойств, исключающих оборотоспособность данных объектов, мож- но указать на их нематериальный характер, неповторяемость, не возможность измерения с помощью вещно-правовых категорий (вес, масса) и др. ; − необоротоспособные объекты по при- знаку неразрывной связи с определенным участником гражданского оборота, например, имущественные права требования выплаты алиментов, возмещения вреда, причиненно- − необоротоспособные объекты по при- знаку неразрывной связи с определенным участником гражданского оборота, например, имущественные права требования выплаты алиментов, возмещения вреда, причиненно- Актуальные проблемы российского права. 2017. № 1 (74) январь 128 Л. В. Борисова О проблеме законодательного регулирования оборотоспособности объектов гражданских прав Л. В. Борисова Л. В. Борисова О проблеме законодательного регулирования оборотоспособности объектов гражданских прав Например, не приобретается право собст- венности на постройку, возведенную с суще- ственным нарушением градостроительных и строительных норм и правил (имеющую неу- странимые нарушения, которые могут повлечь уничтожение постройки, причинение вреда жизни, здоровью человека, повреждение или уничтожение имущества других лиц и др.)5. Такие постройки не могут быть предметом оборота — их запрещено продавать, дарить, сдавать в аренду, совершать другие сделки. В соответствии с ч. 2 ст. 222 ГК РФ самовольные постройки подлежат сносу. Например, сложив- шаяся в настоящее время судебная практика идет по пути отождествления с самовольными постройками самовольно реконструирован- ных объектов независимо от характера рекон- струкции. Реконструкцией признаются работы, затрагивающие конструктивные характери- стики здания и влияющие на его надежность и безопасность (Постановление ФАС ЗСО от 30.10.2006 № Ф04-3743/2006 (26764-А75-39)). го жизни или здоровью, право регрессного требования и др. ; − необоротоспособные объекты в силу закона: запасы материального резерва (п. 3 ст. 4 Федерального закона от 29 декабря 1994 г. № 79 ФЗ «О государственном материальном ре- зерве»); имущество государственной авиации, в том числе органов внутренних дел, а также объекты единой системы организации воздуш- ного движения (ст. 7 Воздушного кодекса РФ от 19.03.1997 № 60-ФЗ); архивные документы, на- ходящиеся в государственной или муниципаль- ной собственности (п. 3—4 ст. 10 Федерального закона от 22 октября 2004 г. № 125-ФЗ «Об ар- хивном деле в Российской Федерации») и др. Необоротоспособными следует считать объекты, назначение или свойства которых признаны законом столь опасными, что пере- ход от одних лиц к другим и установление на них государственной собственности не допустимы. К таким объектам относятся поддельные ле- карства или их незаконные копии (п. 9 ст. 20 Федерального закона «Об обращении лекар- ственных средств» от 12.04.2010 № 61-ФЗ); некачественные и опасные пищевые продук- ты и изделия (ст. 5 Обзор судебной практики по делам, связанным с самовольным строительством, утвержденный Пре- зидиумом Верховного Суда РФ 19.03.2014 // Бюллетень Верховного Суда РФ. 2014. № 6. 6 Подр. см.: О.А. Беляева. Объекты самовольной постройки: по какому пути идет судебная практика // Журнал «Арбитражное правосудие в России» № 6/2008 [Электронный ресурс]: Доступ из справ.- правовой системы «Консультант плюс». д у р уд // р уд 6 Подр. см.: О.А. Беляева. Объекты самовольной постройки: по какому пути идет судебная практика // Журнал «Арбитражное правосудие в России» № 6/2008 [Электронный ресурс]: Доступ из справ.- правовой системы «Консультант плюс». 5 Обзор судебной практики по делам, связанным с самовольным строительством, утвержденный Пре- зидиумом Верховного Суда РФ 19.03.2014 // Бюллетень Верховного Суда РФ. 2014. № 6. 6 По р с О А Бе яева Об е са ово ой ос рой о а о е с еб ая ра а // 7 См.: Определение Верховного суда Российской Федерации от 28 марта 2016 г. № 51-ПЭК16 //[Элек- тронный ресурс]: Доступ из справ.- правовой системы «Консультант плюс». DOI 10.17803/1994-1471.2017.74.1.126-131 24 Федерального закона «О качестве и безопасности пищевых продуктов» от 02.01.2000 № 29-ФЗ); продукция, не соот- ветствующая санитарным нормам и правилам (п. 3 ст. 13 Федерального закона «О санитар- но-эпидемиологическом благополучии насе- ления» от 30.03.1999 № 52-ФЗ); продукты пи- тания, медикаменты, товары бытовой химии и иные товары, по истечении срока годности (п. 4 ст. 5 Закон РФ от 07.02.1992 № 2300-1 «О защите прав потребителей») и др. Правила о самовольной постройке приме- нимы в случаях, когда внутри здания создаются новые помещения, поскольку таковые являют- ся самостоятельными объектами недвижимо- сти с особым гражданско-правовым режимом владения, пользования и распоряжения и др.6 Изымаются из оборота и уничтожаются за счет нарушителя контрафактные товары, эти- кетки, упаковки товаров, на которых размеще- ны незаконно используемый товарный знак или сходное с ним до степени смешения обо- значение (ч.2 ст. 1515 ГК РФ). Например, в не- давно опубликованном определении Верхов- ного Суда Российской Федерации от 28 марта 2016 г. № 51-ПЭК16, суды запретили, а также обязали за свой счет изъять из оборота и унич- тожить товар импортера питьевой воды Vittel. В рамках данного спора ООО «Аквалайф» ввозил воду Vittel на территорию РФ без согласия пра- вообладателя — АОУТ «Нестле Вотерс Фран». По мнению ООО «Аквалайф», предписание су- Не могут относиться к ограниченным в обороте объектам самовольные постройки, фальшивые денежные знаки, контрафактная продукция. Право собственности на данные объекты не приобретается, они подлежат сно- су, изъятию и уничтожению. 5 Обзор судебной практики по делам, связанным с самовольным строительством, утвержденный Пре- зидиумом Верховного Суда РФ 19.03.2014 // Бюллетень Верховного Суда РФ. 2014. № 6. 6 Подр. см.: О.А. Беляева. Объекты самовольной постройки: по какому пути идет судебная практика // Журнал «Арбитражное правосудие в России» № 6/2008 [Электронный ресурс]: Доступ из справ.- правовой системы «Консультант плюс». Актуальные проблемы российского права. 2017. № 1 (74) январь 129 ГРАЖДАНСКОЕ И СЕМЕЙНОЕ ПРАВО К примеру, не может сдаваться в аренду обо- рудование для производства алкогольной продукции (п. 6 ст. 8, п. 1 ст. 26 Федерально- го закона «О государственном регулировании производства и оборота спирта, алкогольной и спиртосодержащей продукции»); не могут быть предметом залога объекты религиозного назначения (п. 5 ст. 21 Федерального закона «О свободе совести и религиозных объединени- ях»); запрещается розничная торговля табач- ной продукцией на ярмарках, выставках, путем развозной и разносной торговли, дистанци- онным способом продажи, с использованием автоматов и иными способами, за исключени- ем развозной торговли в случае, предусмот- ренном частью 2 ст. Актуальные проблемы российского права. 2017. № 1 (74) январь DOI 10.17803/1994-1471.2017.74.1.126-131 19 Федерального закона от 23.02.2013 № 15-ФЗ «Об охране здоровья граждан от воздействия окружающего табач- ного дыма и последствий потребления табака» и др. дов изъять и уничтожить товар неисполнимо. В жалобе заявитель настаивал на том, что выпуск товара в гражданский оборот и его отсутствие у импортера влечет невозможность принятия решения о его уничтожении и изъятии из обо- рота в случае признания его контрафактным. Однако экономическая коллегия ВС РФ, довод импортера ООО «Аквалайф» об отсутствии у него товара отклонила: импортер не предста- вил доказательств, подтверждающих отсутст- вие у него товара и невозможности исполне- ния судебного акта7. В заключении приведем еще одно проти- воречие п. 2 ст. 129 ГК РФ и правил специаль- ного законодательства, которое заключается в том, что ограничение объектов гражданских прав в совершении сделок по специальному разрешению, не охватывает всех видов, преду- смотренных специальным законодательством ограничений, препятствующих их свободному обращению. Таким образом, специальное законода- тельство, предусматривает иные виды ограни- чений оборотоспособности объектов граждан- ских прав, чем противоречит действующему ГК РФ. Для устранения данного противоречия счи- таем необходимым п. 2 ст. 129 ГК РФ изложить более расширительно: вместо слов «по специ- альному разрешению», использовать словосо- четание «с учетом ограничений определенных законом или в установленном им порядке». Так, не согласуются с данным пунктом ГК РФ, сделки с соблюдением предусмотренного законом обязательного порядка (с психотроп- ными веществами, включенными в Список III, оборудованием для производства и изготовле- ния наркотических средств, психотропных ве- ществ (п. 5 ст. 5 и п. 4 ст. 8 Федерального зако- на «О наркотических средствах и психотропных веществах»); драгоценными металлами и кам- нями из Государственного фонда драгоценных металлов и драгоценных камней (п. 32 Поло- жения о Государственном фонде драгоценных металлов и драгоценных камней утв. Поста- новлением Правительства РФ от 27 февраля 2003 г. № 127 «Об утверждении Положения о Государственном фонде драгоценных метал- лов и драгоценных камней Российской Феде- рации») и др. В качестве вывода отметим, что, по наше- му мнению, проблемы законодательного ре- гулирования оборотоспособности объектов гражданских прав связаны с различными от- ступлениями от принципа системности права. Поэтому совершенствование соответствующих норм должно носить последовательный и си- стемный характер, основываться на разрабо- танных в теории гражданского права подходах, а также проверенных временем правилах за- конодательной техники. Сходная ситуация наблюдается с запретом отдельных сделок с некоторыми объектами. Актуальные проблемы российского права. 2017. № 1 (74) январь 130 BORISOVA Liliya Vladimirovna BORISOVA Liliya Vladimirovna PhD in Law, Associate Professor, Financial Uiversity under the Government of the Russian Federation, Associate Professor at the Department of Legal Regulation of Economic Activities grpfa@yandex.ru 125993, Russia, Moscow, Leningradskiy Prospekt, 49 BORISOVA Liliya Vladimirovna PhD in Law, Associate Professor, Financial Uiversity under the Government of the Russian Federation, Associate Professor at the Department of Legal Regulation of Economic Activities grpfa@yandex.ru 125993, Russia, Moscow, Leningradskiy Prospekt, 49 BORISOVA Liliya Vladimirovna PhD in Law, Associate Professor, Financial Uiversity under the Government of the Russian Federation, Associate Professor at the Department of Legal Regulation of Economic Activities grpfa@yandex.ru 125993, Russia, Moscow, Leningradskiy Prospekt, 49 Review. The article highlights and analyses selected issues of legislative regulation of transferability of objects of civil rights. The attention is drawn to the shortcomings of the legislative technique made in the formulation of Art. 129 of the Civil Code of the Russian Federation, its incompleteness and the contradiction with the relevant rules of special legislation. In particular, it is noted that there is some difference in the content of the term “transferability” in this and other legislative acts. The author shows the need to broaden the criteria limiting the transferability of objects of civil rights in transactions by special permission. The author puts forward possible solutions to these problems and contradictions. The article provides some critical remarks on the elimination of the rules on the treatment of seized objects from Art. 129 of the Civil Code, which, due to the Federal law of July 2, 2013 No. 142- FZ On Amendments to Subsection 3, Section 1, Part 1 of the Civil Code of the Russian Federation, are recognized to be limited in conveyance. In connection with this, the author proposes legislative allocation of non-transferable group of objects, which, according to the author, can fall into three categories: non-transferable objects by virtue of their inherent natural properties, indications of the law, or a seamless connection with a specific member of the civilian transactions. The article may be useful for researchers, teachers, graduate students, undergraduates, law students, as well as to all people interested in issues of civil law. Keywords: transferability, the object of civil rights, objects taken out of trade, objects limited in circulation, civil circulation, legal regime, bargain, special legislation, succession. Актуальные проблемы российского права. 2017. № 1 (74) январь BORISOVA Liliya Vladimirovna Keywords: transferability, the object of civil rights, objects taken out of trade, objects limited in circulation, civil circulation, legal regime, bargain, special legislation, succession. БИБЛИОГРАФИЯ 1. Белов В. А. Гражданское право: Общая часть: Учебник. М., 2011. Т. II. 1. Белов В. А. Гражданское право: Общая часть: Учебник. М., 2011. Т. II. 1. Белов В. А. Гражданское право: Общая часть: Учебник. М., 2011. Т. II. 2. Гражданское право: Учебник: В 4 Т. Общая часть / Под ред. Е. А. Суханова. М. : Волтерс Клувер, 2008. Т. 1. 2. Гражданское право: Учебник: В 4 Т. Общая часть / Под ред. Е. А. Суханова. М. : Волтерс Клувер, 2008. Т. 1. 3. Комментарий к Гражданскому кодексу Российской Федерации (части первой) (постатейный) / под ред. О. Н. Садикова. М., 2003. 3. Комментарий к Гражданскому кодексу Российской Федерации (части первой) (постатейный) / под ред. О. Н. Садикова. М., 2003. Беляева О.А. Объекты самовольной постройки: по какому пути идет судебная практика // Журн итражное правосудие в России» № 6/2008. СПС «Консультант плюс». 5. Харитонова Ю.С. КомМ. к ст. 1180 ГК РФ. Научно-практический комментарий к Гражданскому кодек- су Российской Федерации : в 2 т. Т. 2. Ч. 3, 4 ГК РФ / под ред. Т. Е. Абовой, М. М. Богуславского, А. Г. Светла- нова. — Ин-т государства и права РАН. Издательство «Юрайт», 2011. 5. Харитонова Ю.С. КомМ. к ст. 1180 ГК РФ. Научно-практический комментарий к Гражданскому кодек- су Российской Федерации : в 2 т. Т. 2. Ч. 3, 4 ГК РФ / под ред. Т. Е. Абовой, М. М. Богуславского, А. Г. Светла- нова. — Ин-т государства и права РАН. Издательство «Юрайт», 2011. Материал поступил в редакцию 11 октября 2016 г. Материал поступил в редакцию 11 октября 2016 г. REFERENCES (TRANSLITERATION) 1. Belov V. A. Grazhdanskoe pravo: Obshhaja chast’: Uchebnik. M., 2011. T. II. 2. Grazhdanskoe pravo: Uchebnik: V 4 T. Obshhaja chast’ / Pod red. E. A. Suhanova. M. : Volters Kluver, 2008. T. 1. 3. Kommentarij k Grazhdanskomu kodeksu Rossijskoj Federacii (chasti pervoj) (postatejnyj) / pod red. O. N. Sadikova. M., 2003. 4. Beljaeva O.A. Ob#ekty samovol’noj postrojki: po kakomu puti idet sudebnaja praktika // Zhurnal «Arbitrazhnoe pravosudie v Rossii» № 6/2008. SPS «Konsul’tant pljus». 5. Haritonova Ju.S. KomM. k st. 1180 GK RF. Nauchno-prakticheskij kommentarij k Grazhdanskomu kodeksu Rossijskoj Federacii : v 2 t. T. 2. Ch. 3, 4 GK RF / pod red. T. E. Abovoj, M. M. Boguslavskogo, A. G. Svetlanova. — In-t gosudarstva i prava RAN. Izdatel’stvo «Jurajt», 2011. Актуальные проблемы российского права. 2017. № 1 (74) январь 131
https://openalex.org/W3094426228	https://tehj.springeropen.com/track/pdf/10.1186/s43044-020-00104-x	English	null	Long-term follow-up of therapeutic efficacy of everolimus-eluting bioresorbable vascular scaffold in comparison to everolimus-eluting stent in treatment of chronic total occlusion guided by intracoronary imaging	The Egyptian Heart Journal /The Egyptian Heart Journal	2,020	cc-by	8,888	RESEARCH Open Access Long-term follow-up of therapeutic efficacy of everolimus-eluting bioresorbable vascular scaffold in comparison to everolimus-eluting stent in treatment of chronic total occlusion guided by intracoronary imaging Mohammad Abdallah Eltahlawi1 , Abdel-Aziz Fouad Abdel-Aziz2 , Abdel-Salam Sherif1, Khalid Abdel-Azeem Shokry3 and Islam Elsayed Shehata1* Mohammad Abdallah Eltahlawi1 , Abdel-Aziz Fouad Abdel-Aziz2 , Abdel-Salam Sherif1, Khalid Abdel-Azeem Shokry3 and Islam Elsayed Shehata1* Abstract Background: We hypothesized that 1st generation everolimus-eluting bioresorbable vascular scaffold (BVS) stent associated with less complication and less restenosis rate than everolimus-eluting stent (EES) in chronic total occlusion (CTO) recanalization guided by intracoronary imaging. Therefore, we aimed to assess the safety and performance of BVS stent in CTO revascularization in comparison to EES guided by intracoronary imaging. Our prospective comparative cross-sectional study was conducted on 60 CTO patients divided into two groups according to type of stent revascularization: group I (EES group): 40 (66.7%) patients and group II (BVS group): 20 (33.3%) patients. All patients were subjected to history taking, electrocardiogram (ECG), echocardiography, laboratory investigation, stress thallium study to assess viability before revascularization. Revascularization of viable CTO lesion guided by intracoronary imaging using optical coherence tomography (OCT). Then, long-term follow-up over 1 year clinically and by multi-slice CT coronary angiography (MSCT). Our clinical and angiographic endpoints were to detect any clinical or angiographic complications during the follow-up period. laboratory investigation, stress thallium study to assess viability before revascularization. Revascularization of viable CTO lesion guided by intracoronary imaging using optical coherence tomography (OCT). Then, long-term follow-up over 1 year clinically and by multi-slice CT coronary angiography (MSCT). Our clinical and angiographic endpoints were to detect any clinical or angiographic complications during the follow-up period. Results: At 6 months angiographic follow-up, BVS group had not inferior angiographic parameters but without statistically significant difference (p = 0.566). At 12 months follow-up, there was no difference at end points between the two groups (p = 0.476). g p p No differences were found at angiographic or clinical follow-up between BVS and EES. Conclusion: This study shows that 1st generation everolimus-eluting BVS is non-inferior to EES for CTO revascularization. Further studies are needed to clearly state which new smaller footprint BVS, faster reabsorption, magnesium-based less thrombogenicity, and advanced mechanical properties is under development. We cannot dismiss the efficacy and safety of new BVS technology. (Continued on next page) © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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/. * Correspondence: iemshehata@zu.edu.eg * Correspondence: iemshehata@zu.edu.eg 1Department of Cardiology, Faculty of Medicine, Zagazig University, Zagazig 44519, Egypt Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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/. Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 https://doi.org/10.1186/s43044-020-00104-x Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 https://doi.org/10.1186/s43044-020-00104-x (2020) 72:72 Eltahlawi et al. The Egyptian Heart Journal https://doi.org/10.1186/s43044-020-00104-x The Egyptian Heart Journal * Correspondence: iemshehata@zu.edu.eg 1Department of Cardiology, Faculty of Medicine, Zagazig University, Zagazig 44519, Egypt Full list of author information is available at the end of the article Sample size estimation p Assuming a frequency of 2.3% of CTO in controls and 37.4% in cases, with 80% power and 95% CI, the estimated sample was 60 patients (OPEN-EPI-Info version 6). Study site Our study was conducted at the cardiology department of our hospitals. Inclusion criteria Our study included 60 true or probable CTO patients in one or more of the coronary arteries with persistence of severe symptoms (e.g., typical ischemic chest pain, An- gina equivalent) despite maximum medical therapy (e.g., nitrate, anti-platelets, beta-blockers, angiotensin convert- ing enzyme inhibitors, and statins) with evidence of vi- able myocardium confirmed by stress myocardial thallium study in the territory of CTO vessels. Tools and instruments Resting ECG, laboratory for analysis of cardiac troponin I, fasting blood sugar, lipid profile [total cholesterol, tri- glycerides (TG), HDL and LDL], serum Creatinine, Creatinine clearance, C-Reactive Protein (CRP), trans- thoracic echocardiography (TTE), cardiac nuclear stress thallium study, coronary angiography, percutaneous cor- onary intervention (PCI) and optical coherence Time frame Patients were recruited for 24 months from January 2017 to January 2019. Page 2 of 13 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 (Continued from previous page) Trial registration: ZU-IRB#2498/3-12-2016 Registered 3 December 2016, email: IRB_123@medicine.zu.edu.eg Keywords: Bioresorbable vascular stents, Chronic total occlusion, Coronary artery disease, Drug-eluting stent, Intracoronary imaging, Multi-slice CT coronary angiography p p g Trial registration: ZU-IRB#2498/3-12-2016 Registered 3 December 2016, email: IRB_123@medicine.zu.edu.eg Keywords: Bioresorbable vascular stents, Chronic total occlusion, Coronary artery disease, Drug-eluting stent, Intracoronary imaging, Multi-slice CT coronary angiography Exclusion criteria We hypothesized that BVS associated with less com- plication and less restenosis rate than EES in CTO re- canalization guided by intracoronary imaging. Therefore, we aimed to assess the safety and performance of BVS stent in CTO revascularization in comparison to EES guided by intracoronary imaging. Patient refusal, extremely calcified tortuous lesions re- sistant or non-dilatable after trials of plaque modifica- tion by cutting balloon or rotational atherectomy and confirmed by OCT evaluation, true bifurcated lesions with side branch > 2.5mm, reference vessel diameter < 2.5 mm or > 4.5 mm (out of the BVS measures), and complications treated with metallic stents. Study population The study group comprised 60 patients with true chronic total occlusion (CTO) diagnosed by coronary angiography whom scheduled for revascularization of CTO lesion in our hospital who fulfilled the inclusion criteria and followed up over 24 months. Background according to type of stent revascularization: group I (EES group): 40 (66.7%) patients and group II (BVS group): 20 (33.3%) patients. Coronary artery disease (CAD) has been the main cause of death in the world [1, 2]. The incidence of CAD is in- creasing yearly more in younger patients [3, 4]. Coronary angiography remains the clinical gold standard for diag- nosis of coronary artery disease [5]. Currently drug- eluting stents (DES) used for revascularization of CAD with significant stenosis (> 70%) [6]. Drug carriers of DES are mainly polymer coatings, which are developed to carry enough anti-proliferation drug dosage and can perfectly control the degradation, penetration and re- lease of everolimus or other drugs. The newly invented bioabsorbable vascular stent (BVS) have a number of benefits [7, 8]. First, BVS opens the occlusion of coron- ary artery. Second, after its absorption, BVS can restore endothelial function and normal vasomotion. Third, avoid a “full metal jacket,” precluding future coronary artery bypass graft (CABG). Previous studies have been conducted to compare the efficacy of BVS with DES but the outcomes were inconsistent and remain to be identi- fied [9–12]. Methods Also we excluded patients with moderate-to-severe valvular heart disease, prosthetic heart valve, bundle branches block (LBBB or RBBB), atrial fibrillation (AF), paced rhythm, an atrioventricular block, restrictive, hypertrophic, or dilated cardiomyopathies, congenital heart disease, coronary artery ectasia, previous history of myocardial infarction, uncontrolled hypertension, hyper- thyroidism, hypothyroidism, malignancy, or pulmonary, hepatic, renal, or hematological disorders. Study methodology – All patients were subjected to the following: Full history was taken with special emphasis on history of risk factors for ischemic heart disease (IHD) and family history of premature IHD, including the following: i. Hypertension was defined as the level of office systolic BP values are ≥140 mmHg and/or diastolic BP values are ≥90 mmHg which is equivalent to a 24-h ambulatory blood pres- sure (ABPM) average of ≥130/80 mmHg or an home blood pressure monitoring (HBPM) average of ≥135/85 mmHg in younger, middle-aged, and older people [13]. ii. Diabetes mellitus was diagnosed based on presence of two criteria from the following: Laboratory investigations including cardiac biomarkers: blood samples for troponin were collected, on presentation. Troponin I was considered positive if it exceeds the 99th percentile of normal reference (above 0.01 ng/ mL), fasting blood sugar on admission, lipid profile: fasting 12 h including total cholesterol, triglycerides (TG), HDL and LDL, serum creatinine and creatinine clearance, and CBC. Fasting blood sugar (FBS) ≥126 mg/dL (7.0 mmol/L) Two-hour post-prandial blood sugar (2 h- PPBS) ≥200 mg/dL (11.1 mmol/L) during oral glucose tolerance test (OGTT) (75 g) HbA1C ≥6.5% (48 mmol/mol) HbA1C ≥6.5% (48 mmol/mol) Random blood sugar ≥200 mg/dL (11.1 mmol/L) [14]. Myocardial perfusion index (MPI): stress myocardial Thallium study done to all cases of CTO to detect myocardial viability before revascularization. iii. Dyslipidemia defined in CTO (very high risk patients) if LDL-C more than 55 mg/dL (1.4 mmol/L) [15]. Coronary angiography was performed using the Judkins method. Stents were implanted using a routine method. Procedure success indicated residual stenosis < 20%, TIMI flow grade III, and no acute complication (death, myocardial infarction, emergency CABG), and no major adverse cardiac events [(cardiac death, myocardial infarction, target vessel revascularization (TVR)] in hospital. Clinical follow-up was performed at 6 and 12 months. Coronary angiography was performed using the Judkins method. Stents were implanted using a routine method. Procedure success indicated residual stenosis < 20%, TIMI flow grade III, and no acute complication (death, myocardial infarction, emergency CABG), and no major adverse cardiac events [(cardiac death, myocardial infarction, target vessel revascularization (TVR)] in hospital. Clinical follow-up was performed at 6 and 12 months. iv. Study design The present prospective comparative cross-sectional study included 60 CTO patients divided into two groups Page 3 of 13 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 tomography (OCT), and multi-slice CT coronary angiog- raphy (MSCT). with other cardiac disorders as LV hypertrophy or previous MI. Transthoracic echocardiography (Fig. 1) was performed using a Vivid 9 system (GE Healthcare, Little Chalfont, UK) apparatus on admission for assessment the heart including LV ejection fraction, wall motion score index, left ventricular end-systolic volume (ESV), end- diastolic volume (EDV), and LV ejection fraction (EF) were measured from apical two and four chamber views using modified Simpson’s biplane method and the mean of the two readings was then taken. End-systole was defined as the frame with the smallest cavity area and end diastole as the frame with the largest LV cavity area. The EF was then calculated using the following formula for each view: EF (%) = [(EDV −ESV)/EDV] × 100 [18]. Transthoracic echocardiography (Fig. 1) was performed using a Vivid 9 system (GE Healthcare, Little Chalfont, UK) apparatus on admission for assessment the heart including LV ejection fraction, wall motion score index, left ventricular end-systolic volume (ESV), end- diastolic volume (EDV), and LV ejection fraction (EF) were measured from apical two and four chamber views using modified Simpson’s biplane method and the mean of the two readings was then taken. End-systole was defined as the frame with the smallest cavity area and end diastole as the frame with the largest LV cavity area. The EF was then calculated using the following formula for each view: EF (%) = [(EDV −ESV)/EDV] × 100 [18]. Study methodology Current smoker was defined according to the National Health Interview Survey (NHIS) as a person who reports currently smoking tobacco every day (i.e., daily smoker) or on some days (nondaily smoker) [16]. v. Family history of CAD was defined as family history of early coronary artery disease in the first-degree relatives, male < 55 years and fe- males < 65 years [17]. Thorough physical examination including pulse, heart rate, blood pressure (systolic and diastolic), neck veins, edema of lower limbs, abdominal examination, chest examinations, and cardiac examination including inspection, palpation, and auscultation. – The culprit coronary lesion was clearly identified by a combination of ECG and coronary angiography. By complete occlusion of target artery TIMI 0 more than 3 months. – Lesion is estimated by J score which calculated from 5 points described by Christopoulos G et al. (2015) combined 5 baseline clinical and angiographic CTO parameter one point is given for each of the following factor that are associated with lower probability of successful guide wire crossing: blunt stump, calcification, lesion bending > 45°, occlusion length > 20 mm A resting baseline 12-lead ECG was carried out on admission, post-PCI and at regular follow-up periods (1st, 6th, and 12th months) at a paper speed of 25 mm/s and amplification of 10 mm/ mv. ECG was analyzed for: ST segment deviation, Q wave at number of leads with interpreted to site of old MI. Comparison with a previous ECG, when available, was valuable especially in patients Page 4 of 13 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Fig. 1 Method of calculation of biplane Simpson method. LVEDV: Left ventricular end-diastolic volume, LVESV: Left ventricular end-systolic volume, A4C: Apical 4-chamber view, A2C: Apical 2-chamber view Fig. 1 Method of calculation of biplane Simpson method. LVEDV: Left ventricular end-diastolic volume, LVESV: Left ventricular end-systolic volume, A4C: Apical 4-chamber view, A2C: Apical 2-chamber view – During procedure activated clotting time was maintained above 300 ms. – During procedure activated clotting time was maintained above 300 ms. – During procedure activated clotting time was maintained above 300 ms. and prior failed attempt to revascularize the CTO and classified into: J-CTO = 0 is easy, J-CTO = 1 is intermediate, J-CTO = 2 is difficult, and J-CTO ≥3 is very difficult [19]. Results 9- All BVS was deployed after good preparation using rotablation for highly fibrotic lesions and assessment of well deployment by OCT study (Fig. 3). 9- All BVS was deployed after good preparation using rotablation for highly fibrotic lesions and assessment of well deployment by OCT study (Fig. 3). 9- All BVS was deployed after good preparation using rotablation for highly fibrotic lesions and assessment of well deployment by OCT study (Fig. 3). Study methodology and prior failed attempt to revascularize the CTO and classified into: J-CTO = 0 is easy, J-CTO = 1 is intermediate, J-CTO = 2 is difficult, and J-CTO ≥3 is very difficult [19]. 1- The procedure was done most frequently by antegrade approach in 75% in both groups and by retrograde approach in 25% in both groups if failed trial by antegrade approach. – Quantitative coronary angiography (QCA) was performed in the first angiography by two independent investigators who were blinded to the results. 2- Crossing the occluded segment by guiding wire then dilatation initially by small balloon (1 mm) – All patients are loaded by dual anti-platelet therapy by Aspirin (300 mg) and Clopidogrel (600 mg once) therapy. 3- After nitroglycerin infusion up to (600 mcg), vessel size and lesion length were determined by quantitative coronary angiography (QCA). – All clinical, laboratory, and coronary angiographic data were evaluated by 2 independent investigators who were not involved in the angiographic procedures. 4- Pre-dilatation of entire lesion by correct balloon size. Technical steps for percutaneous coronary intervention (PCI) 5- Optical coherence tomography (OCT) (Fig. 3): FD-OCT (C7-XR system) using dedicated software (Medis Qlvus 3.0 with OCT quantification software) was used to analyze morphological and ana- tomical characteristics before stent im- plantation in all cases. 1- Preprocedural – PCI strategy was decided to each case according to revision of coronary angiography and clinical examination (contralateral injection, guiding catheter choice, antegrade, or retro-grade approach). 6- Optimal pre-dilatation by either cutting or noncompliant balloon to decrease risk of under expansion of stent area. 2- Intraprocedural steps Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Page 5 of 13 7- Stent implantation according to OCT measurement either 1st generation BVS or 3rd generation EES. 7- Stent implantation according to OCT measurement either 1st generation BVS or 3rd generation EES. 7- Stent implantation according to OCT measurement either 1st generation BVS or 3rd generation EES. Correlations between variables were analyzed using the Pearson correlation coefficient. All data were analyzed using SPSS software version 22 (SPSS, Inc. Chicago, IL, USA). 8- BVS implantation by increasing pressure by 2 ATM every 5 s up to 12–14 ATM with overlapping by only 1–2 mm. 8- BVS implantation by increasing pressure by 2 ATM every 5 s up to 12–14 ATM with overlapping by only 1–2 mm. Echocardiography data on admission (Table 3) No significant difference was found between both group regarding EF (p = 0.652). Echocardiography data on admission (Table 3) No significant difference was found between both groups regarding EF (p = 0.652). No significant difference was found between both groups regarding EF (p = 0.652). Diastolic dysfunction is present in 97% in EES group and 100% in BVS group (p = 0.476). Statistical methods As regards to wall motion, there was no wall motion abnormalities in 57.5% in EES group and in 50% in BVS group, anterior hypokinesia 32.5% in EES group, and 35% in BVS group and inferior hypokinesia 10% in both groups and 5% lateral wall hypokinesia in BVS group. The statistical analysis was conducted following the principles as specified in International Council for Harmonization (ICH) Topic E9 (ICH1998). For continu- ous measurements, data were presented using mean, median, standard deviation, minimum, and maximum, and for the categorical measurements, data were pre- sented using absolute/relative frequencies and percent- ages. To find the association between categorical variables, a chi-squared test or Fisher exact test was used. For all statistical analyses, a p value of less than or equal to 0.05 was considered to indicate a significant difference. Clinical presentation on admission (Table 2) 3- Post-procedural – All patients were treated by dual anti- platelet therapy after the procedure. Patient symptoms were assessed according to chest pain and dyspnea. There was no significant difference be- tween both groups regarding symptoms (p >0.05). p py p Follow up: Intraprocedural: acute procedural success was achieved by TIMI flow 3 and stenosis less than 20% without any complications. Intraprocedural complications were recorded. 1st, 6th and 12th month: Clinical follow up at 1st, 6th, and 12th month done for chest pain and hospitalization and major adverse cardiovascular events (MACE). Any death from unknown cause was considered cardiac. Evidence of myocardial damage as CK > 3 times of upper limit of normal and troponin > 5 times upper limit of normal. All patients underwent multi-slice CT coronary angiog- raphy (MSCT) after 6 months and after 12 months to detect any in-stent restenosis. Follow up: Intraprocedural: acute procedural success was achieved by TIMI flow 3 and stenosis less than 20% without any complications. Intraprocedural complications were recorded. 1st, 6th and 12th month: Clinical follow up at 1st, 6th, and 12th month done for chest pain and hospitalization and major adverse cardiovascular events (MACE). Any death from unknown cause was considered cardiac. Evidence of myocardial damage as CK > 3 times of upper limit of normal and troponin > 5 times upper limit of normal. All patients underwent multi-slice CT coronary angiog- raphy (MSCT) after 6 months and after 12 months to detect any in-stent restenosis. Follow up: Regarding clinical examination, we found no signifi- cant difference between both group in respect to BP, pulse, and murmur (p > 0.05). Baseline clinical characteristics Demographic data (Table 1) Baseline clinical characteristics showed approximately 90% of study were male and baseline clinical characteris- tics were similar between 2 groups. Mean age of patients was 57 ± 6.98 in DES group and 56.15 ± 9.25 in BVS group (p = 0.568). 10- If we need post-dilatation, this would be done using shorter NC balloon at nominal pressure with maximal in- crease of balloon size 0.5 mm above stent size. There was no significant difference between both groups regarding age, sex, DM, hyperlipidemia, HTN, history of MI, family history of premature CAD, and previous PCI or CABG (p > 0.05). 11- OCT: OCT study done with visual assessment during procedure to entire segment and NC balloon post- dilatation if any residual stenosis appeared. Laboratory investigations on admission (Table 3) There was no significant difference between both groups as regard to CBC and serum creatinine (p > 0.05). No significant difference was found between 2 groups Laboratory investigations on admission (Table 3) There was no significant difference between both groups as regard to CBC and serum creatinine (p > 0.05). There was no significant difference between both groups as regard to CBC and serum creatinine (p > 0.05). No significant difference was found between 2 groups as regard to ECG which reveal normal in 42% in EES group and 35% in BVS group (p = 0.202). Myocardial perfusion index on admission (MPI) (Table 3) There was no significant difference between both groups regarding myocardial perfusion index which revealed vi- able myocardium in 32.5% in EES group and 20% in BVS group (p = 0.84). Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Page 6 of 13 Table 1 Demographic data History (risk factors) EES group BVS group Test p value Sig. No. = 40 No. = 20 Age Mean ± SD 57.38 ± 6.98 56.15 ± 9.25 0.574b 0.568 NS Range 45–75 34–80 Sex Male 37 (92.5 %) 19 (95%) 0.463b 0.457 NS Female 5 (12.5 %) 1 (5%) DM Negative 19 (47.5%) 8 (40.0%) 0.881a 0.459 NS Positive 21 (52.5) 12 (60.0%) Hyperlipidemia Negative 5 (12.5%) 0 (0.0%) 2.727a 0.099 NS Positive 35 (87.5%) 20 (100.0%) HTN Negative 17 (42.5%) 6 (30.0%) 0.881a 0.348 NS Positive 23 (57.5%) 14 (70.0%) History of MI Negative 24 (60.0%) 14 (70.0%) 0.574a 0.449 NS Positive 16 (40.0%) 6 (30.0%) Family history of premature CAD Negative 31 (77.5%) 17 (85.0%) 0.469a 0.494 NS Positive 9 (22.5%) 3 (15.0%) Previous PCI, CABG Negative 27 (67.5%) 17 (85.0%) 2.088a 0.148 NS Positive 13 (32.5%) 3 (15.0%) NS non-significant, S significant, HTN hypertension, MI myocardial infraction, CAD coronary artery disease, PCI percutaneous coronary intervention, CABG coronary artery bypass graft aChi-square test bIndependent t test NS non-significant, S significant, HTN hypertension, MI myocardial infraction, CAD coronary artery disease, PCI percutaneous coronary intervention, CABG coronary artery bypass graft aChi-square test bIndependent t test Angiographic and procedure characteristics Intraprocedural and post-procedural aspect during intervention (Table 4 and Fig. 2) Angiographic and procedure characteristics Intraprocedural and post-procedural aspect during intervention (Table 4 and Fig. 2) Baseline lesion data presented in Table 4 revealed LAD and RCA were the most frequent index lesion in total study population and each presents 45% of total study population, while LCX presents 10%. y p p p The procedure was done most frequently by antegrade approach in 75% in both groups and by retrograde ap- proach in 25% in both groups. Retrograde approach was y The procedure was done most frequently by antegrade approach in 75% in both groups and by retrograde ap- proach in 25% in both groups. Retrograde approach was Baseline lesion data presented in Table 4 revealed LAD and RCA were the most frequent index lesion Table 2 Clinical presentation on admission Symptoms and signs EES group BVS group Test p value Sig. No. BP blood pressure, SBP systolic blood pressure, DBP diastolic blood pressure, MR mitral regurgitation, NS non-significant, S significant aChi-square test Myocardial perfusion index on admission (MPI) (Table 3) 5 (12.5%) 1 (5.0%) LAHB 0 (0.0%) 3 (15.0%) NAD 17 (42.5%) 7 (35.0%) Echo EF (%) Mean ± SD 57.55 ± 7.55 56.45 ± 11.11 0.453b 0.652 NS Range 45–73 40–85 DD (grade) I 1 (2.5%) 0 (0.0%) 0.508a 0.476 NS II 39 (97.5%) 20 (100.0%) Wall motion No 23 (57.5%) 10 (50.0%) 2.161a 0.540 NS Anterior 13 (32.5%) 7 (35.0%) Inferior 4 (10.0%) 2 (10.0%) Lateral 0 (0.0%) 1 (5.0%) MPI Viable 13 (32.5%) 4 (20%) 2.063 0.840 NS Ischemia anterior 5 (12.5%) 4 (20%) Ischemia inferior 6 (15%) 5 (25%) Ischemia lateral 2 (5%) 1 (5%) Mixed scar anterior 7 (17.5%) 3 (15%) Mixed scar inferior 7 (17.5%) 3 (15%) aChi-square test bIndependent t test, NS non-significant, S significant, CBC complete blood count, DD diastolic dysfunction, ECG electrogardiogram Table 3 Lab, echocardiographic data, and myocardial perfusion index (MPI) on admission i square test dependent t test, NS non-significant, S significant, CBC complete blood count, DD diastolic dysfunction, ECG electrogardiogram C squa e test bIndependent t test, NS non-significant, S significant, CBC complete blood count, DD diastolic dysfunction, ECG electrog simple balloon inflation without progressing to cardiac tamponade. failed to cross CTO site in another 5 (4.0%) patients; those 5 patients had another trial of successful antegrade approach. There were no significant differences in re- spect to angiographic characteristics of the lesion be- tween success and failure groups. Concerning in-hospital outcome in the current study, there was no reported mortality during hospitalization period, Q wave myocardial infarction developed in 1.6% of study population and non Q wave myocardial infarc- tion developed in 2.4%. Evaluation of index lesion was done by J score which revealed no significant difference between both groups (p = 0.022) in J score. Post-dilatation was done in 95% of DES group and 100% of BVS group with no significant different between both groups (p = 0.309). Myocardial perfusion index on admission (MPI) (Table 3) = 40 No. = 20 Chest pain Negative 2 (5%) 0 (0%) 1.034 0.309 NS Positive 38 (95%) 20 (100%) Dyspnea Negative 29 (72.5%) 17 (85%) 1.165 0.281 NS Positive 11 (27.5%) 3 (15%) BP Normal 40 (100%) 20 (100%) – – – SBP Mean ± SD 112.98 ± 3.5 114.67 ± 3.22 1.809 0.076 NS Range 100–120 100–120 DBP Mean ± SD 74.52 ± 1.7 74.80 ± 1.34 0.643 0.523 NS Range 70–80 70–80 Pulse Mean ± SD 74.05 ± 5.81 72.45 ± 4.97 1.053b 0.297 NS Range 60–86 65–76 Murmur No 29 (72.5%) 16 (80.0%) 0.400a 0.527 NS MR 11 (27.5%) 4 (20.0%) BP blood pressure, SBP systolic blood pressure, DBP diastolic blood pressure, MR mitral regurgitation, NS non-significant, S significant aChi-square test bIndependent t test Table 2 Clinical presentation on admission Table 2 Clinical presentation on admission Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Page 7 of 13 Table 3 Lab, echocardiographic data, and myocardial perfusion index (MPI) on admission EES group BVS group Test p value Sig. No. = 40 No. = 20 Lab Hemoglobin Mean ± SD 13.5 ± 0.68 13.2 ± 0.89 1.448b 0.153 NS Range 12–14 12–14 Creatinine Mean ± SD 0.97 ± 0.13 0.99–0.08 −0.639b 0.525 NS Range 0.4 – 1.2 0.8–1.2 ECG NAD 17 (42.5%) 7 (35.0%) 7.265a 0.202 NS Poor R wave progression 3 (7.5%) 2 (10.0%) Q wave and inf. 7 (17.5%) 4 (20.0%) Q wave and ant. 8 (20.0%) 3 (15.0%) T inversion ant. Angiographic and procedure characteristics Regarding angiographic procedure in current study, we found that occluded stent just occurred in 2 patients (10%) of BVS group and 4 patients (10%) of EES group at 6 months follow-up without significant difference be- tween both groups. This large percent can be explained by our small sample size. Unstable angina (UA) requiring hospitalization (Table 5) There was no significant difference between both groups as regard unstable angina requiring hospitalization at 6 months and 1 year follow-up (p = 0.566 and 0.476 respectively). Our study in comparable with primary end point of absorb III trials which showed target lesion failure (TLF) at 12 months, this trial included 2008 patients (revascu- larization by BVS was 1322 and revascularization by DES 686) which showed TLF was 7.8% in BVS group and 6.1% in DES group. So absorb III trials provides ad- equate result of non-inferiority of BVS against DES in treatment of CTO lesions [22]. Baseline clinical characteristics Baseline clinical characteristics Regarding the age of patients in the current study ranged from 34 to 80 years, with mean age of (58 ± 8.6) years. The mean age of the sample in the present study is com- parable to the mean age of patients enrolled in study by Hoye et al. (2005) (59.8 ± 11.1 years); however, Morino et al. (2010) reported a higher mean age in his study on outcomes of PCI in patients with CTOs (66.9 ± 11 years) [20, 21]. Mortality during the follow-up period (Table 5) Fol- low-up of all study population up to 1 year revealed no mortality reported in any group. Non-fatal MI (Table 5) During the follow-up period, there was no reported MI in any group at the 1st and 6th month of follow up. However, there was one MI case in EES group at 1 year follow up while none in BVS group (p = 0.476). The baseline characteristics and risks factors were comparable between the two study groups. Clinical outcome and follow-up All MACE results No significant difference was found in stent size be- tween both groups (p = 0.219). Post-procedural follow-up (Table 5) All study popula- tion of both groups was on dual anti-platelet for at least 1 year post-procedural. TIMI III flow was achieved in Coronary perforation occurred only in one patient (0.05%) of BVS group and was managed successfully by Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Page 8 of 13 Table 4 Intraprocedural and post-procedural aspect during intervention PCI DES group Absorb group Test p value Sig. No. = 40 No. = 20 Artery LAD 18 (45.0%) 9 (45.0%) 0.000a 1.000 NS RCA 18 (45.0%) 9 (45.0%) LCX 4 (10.0%) 2 (10.0%) Antegrade or retrograde Antegrade 30 (75.0%) 15 (75.0%) 0.000a 1.000 NS Retrograde 10 (25.0%) 5 (25.0%) J score Median (IQR) 3 (3 – 4) 3 (3–4) −0.679c 0.497 NS Range 3–4 2–4 Pre-dilatation Done 40 (100.0%) 20 (100.0%) – – – Post-dilatation Not done 2 (5.0%) 0 (0.0%) 1.034a 0.309 NS Done 38 (95.0%) 20 (100.0%) Stent length Mean ± SD 32.53 ± 9.46 29.40 ± 8.61 1.242b 0.219 NS Range 15–48 18–38 DAPT Given 40 (100.0%) 20 (100.0%) – – – TIMI flow intraprocedure II 2 (5.0%) 0 (0.0%) 1.034a 0.309 NS III 38 (95.0%) 20 (100.0%) Coronary perforation occurred 0 (0%) 1 (0.05%) 0.000a 1.000 NS NS non-significant, S significant aChi-square test bIndependent t test c h Table 4 Intraprocedural and post-procedural aspect during intervention 95% in DES group and 100% of BVS group with reveal no significant difference (p = 0.309). The CT Coronary angiography results Multi-slice CT coronary angiography (MSCT) (Table 5 and Fig. 3) No significant difference was found be- tween both groups at 6 months follow-up by MSCT which revealed patent stent in 90% and 10% occluded stent in both groups (p = 1.000). Page 9 of 13 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Fig. 2 a Coronary angiography of CTO of osteal LAD with J-CTO score complexity = 2. b Trial of antegrade approach c Successful retrograde approach. d Successful revascularization of total LAD by DES Fig. 2 a Coronary angiography of CTO of osteal LAD with J-CTO score complexity = 2. b Trial of antegrade approach c Successful retrograde approach. d Successful revascularization of total LAD by DES Absorb-CTO-registry which included 35 patients with true CTO lesion clearly showed that BVS use in CTO in feasible with good mid-term efficacy and 6 months follow-up by multi-slice computed tomography identifies only 2 cases scaffold re-occlusion without any major ad- verse event. Safety and feasibility of BVS implantation in CTO has been confirmed in this registry [23]. These re- sults were matched with our study results. gained popularity recently with increased success rates as shown in several case reports and small series of se- lected patients [25]. Retrograde approach requires good special devices, such as micro catheters, dedicated guide wires, shorter guide catheters, or longer length guide wires, and should be performed with experienced operators and support staff. Another Ghost-CTO-registry which included 21 pa- tients who had CTO lesions which treated by BVS and followed up after 1 year by OCT showed overall favor- able vascular response and healing profile [24]. These re- sults are also comparable with our study. Retrograde approach was successful in 15 (25%) pa- tients, and was failed to cross CTO site in another 5 (4.0%) patients, those 5 patients had another trial of suc- cessful antegrade approach. There were no significant differences in respect to angiographic characteristics of the lesion between success and failure groups. In our study, the lesion location of CTOs were in left anterior descending artery (LAD) in 45% of cases, right coronary artery (RCA) in 45% of cases, and left circum- flex artery (LCX) in 10% of cases. The CT Coronary angiography results However, the conventional factors such as severe calci- fication, severe tortuosity, significant side branch at the CTO site and CTO length, which are shown to be inde- pendent predictors of successful procedure via antegrade approach in various studies [26] are not shown to have any significant impact on outcomes during retrograde recanalization in our study. In the current study, all patients had initial attempt of wiring through antegrade approach, if antegrade ap- proach was failed, then retrograde approach was tried. Retrograde approach has been described initially via the bypass grafts and more recently the use of septal col- laterals has been described to be safe and efficient. Retrograde approach for recanalization of CTO has There was no difference between both groups possibly due to that conventional factors are not important when the retrograde techniques are applied, as the wire Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Page 10 of 13 Table 5 Post-procedural follo-up Table 5 Post-procedural follo-up EES group BVS group χ2 test p value Sig. No. = 40 No. = 20 Mortality 1 month Negative 40 (100%) 20 (100%) – – – Positive 0 (0%) 0 (0%) 6 months Negative 40 (100%) 20 (100%) – – – Positive 0 (0%) 0 (0%) 1 year Negative 40 (100%) 20 (100%) – – – Positive 0 (0%) 0 (0%) Non-fatal MI 1 month Negative 40 (100%) 20 (100%) – – – Positive 0 (0%) 0 (0%) 6 months Negative 40 (100%) 20 (100%) – – – Positive 0 (0%) 0 (0%) 1 year Negative 39 (97.5%) 20 (100%) 0.508 0.476 NS Positive 1 (2.5%) 0 (0%) UA requiring hospitalization 1 month Negative 40 (100%) 20 (100%) – – – Positive 0 (0%) 0 (0%) 6 months Negative 25 (62.5%) 14 (70%) 0.330 0.566 NS Positive 15 (37.5%) 6 (30.0%) 1 year Negative 39 (97.5%) 20 (100%) 0.508 0.476 NS Positive 1 (2.5%) 0 (0%) Multi-slice CT (MSCT) 6 months Patent 36 (90%) 18 (90%) 0.000 1.000 NS Occluded 4 (10%) 2 (10%) 1 year Patent 36 (90%) 18 (90%) 0.000 1.000 NS Occluded 4 (10%) 2 (10%) MI myocardial infarction, UA unstable angina Fig. 3 First panel from the Lt. side: Coronary angiography of CTO of mid RCA with J-CTO score complexity = 2. Second panel: Post-BVS implantation of 3 overlapped BVS Absorb (3.0 × 28; 3.5 × 28, and 3.5 × 28 mm (84 mm length). The CT Coronary angiography results Third panel: OCT Post-BVS implantation. Fourth panel: 6 months MSCT follow-up. Fifth panel: OCT 12 months post-BVS Fig. 3 First panel from the Lt. side: Coronary angiography of CTO of mid RCA with J-CTO score complexity = 2. Second panel: Post-BVS implantation of 3 overlapped BVS Absorb (3.0 × 28; 3.5 × 28, and 3.5 × 28 mm (84 mm length). Third panel: OCT Post-BVS implantation. Fourth panel: 6 months MSCT follow-up. Fifth panel: OCT 12 months post-BVS Page 11 of 13 Page 11 of 13 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 no significance between 2 groups about MACE at 12 months follow-up. manipulation is done in retrograde fashion to gain ac- cess to either true or false lumen and to complete the procedure with adjunctive techniques. Our study shows non inferiority of BVS stent in CTO comparable with EES which matched with absorb III trial. However, our study of small population size limit data to show advantage of BVS on DES. Regarding procedure related complication in the current study, there was no reported cases of mortality or stent thrombosis, this might be due to operators ex- perience and use of intense anti-platelet regimen and limited number of patients. The knowledge that PCI does not prevent MI or pro- long life in stable or stabilized coronary disease is well established. This was reinforced by the COURAGE and OAT trials in which PCI did not provide incremental ben- efits in hard end points such as MI or mortality above contemporary medical management. However, because COURAGE did not address CTOs specifically and OAT addressed only subacute post-MI occlusions, the general- ized ability of these trials to CTO is not clear [29] One of the most fatal complications during CTO inter- vention is coronary perforation and tamponade [21]. In spite of angiographic coronary perforation was fre- quently observed during CTO treatment, so evaluating the clinical significance of angiographic perforation is critically important, and goal must be to avoid cardiac tamponade. Furthermore, the COURAGE nuclear substudy dem- onstrated event reduction in patient with moderate to severe ischemia. The CTO populations are often enriched with patients with high ischemic burden who may likely benefit from recanalization [30]. In the current study, coronary perforation occurred only in one patient (0.05%) of BVS group and was man- aged successfully by simple balloon inflation without progressing to cardiac tamponade. Clinical outcome and follow-up Concerning in-hospital outcome in the current study, there was no reported mortality during hospitalization period, Q wave myocardial infarction developed in 1.6% of study population and non Q wave myocardial infarc- tion developed in 2.4%. Limitations of the study This study was limited by its small sample size in pa- tients from a two medical centers. Second generation BVS was not used for comparison as it is still under de- velopment. We need longer follow up to detect the out- come of BVS. Clinical implication Close figures were reported by Lemos et al. (2004) who reported no death, TLR, TVR or Q wave MI, but incidence of 7.4% of non Q wave MI [27]. ○We believe that 1st generation everolimus-eluting bioresorbable vascular scaffold (BVS) stent associated with less complication and less restenosis rate than everolimus-eluting stent (EES) in chronic total occlu- sion (CTO) recanalization guided by intracoronary imaging. After the introduction of DES, several studies demon- strated that DES markedly reduce the incidence of angiographic restenosis and repeat revascularization in selected patients with relatively noncomplex lesions. In patients with CTO lesions, there are several trials com- paring the efficacy of DES with BMS but studies com- paring individual DESs against BVS are limited [28]. ○Further follow-up data should be done on a large scale in order to provide evidence for how to follow-up and treat those patients. Regarding 12-month clinical follow-up in our study, no mortality occurred in all study population in both arms, and at 6 months follow-up unstable angina oc- curred in 37% with EES and 30% in BVS stent group which agreement with other previous studies from mul- tiple randomized controlled trials (RCT) while revealing The CT Coronary angiography results Similar figure was reported by Morino et al. (2010) and they reported incidence of 0.4%. These figures are considered low compared with the previous reports (a range of 0.8% to 1.9%) [21]. Coronary BVS are attractive therapeutic option in inter- ventional cardiology; BVS backbone is made of poly- lactoide and coated by thin layer of poly-dilactrole. It re- leases everolimus and is fully degraded to H2O and CO2 in 2–3 years. BVS seems to offer several theoretical advan- tage over metallic stent, as it gives temporary mechanical support of vessel wall; therefore, long-term endothelial function and structure are not affected, and a possible fu- ture surgical revascularization is not compromised [31]. Potential hypotheses to explain low incidence of car- diac tamponade are adequate hemostatic procedures as needed, reduction of prolonged and aggressive antegrade guide wire manipulation. Thanks to the introduction of retrograde approaches. Although a careful evaluation is required, procedural continuation could be considered with angiographic coronary perforation. Conclusion In light of our findings, we conclude that our study showed that 1st generation everolimus-eluting BVS is non-inferior to EES for CTO revascularization. Further studies are needed to clearly state which new smaller Page 12 of 13 Page 12 of 13 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Eltahlawi et al. The Egyptian Heart Journal (2020) 72:72 Received: 5 July 2020 Accepted: 5 October 2020 Received: 5 July 2020 Accepted: 5 October 2020 Funding The study was performed with no funding sources. Received: 5 July 2020 Accepted: 5 October 2020 footprint BVS, faster reabsorption, magnesium-based less thrombogenicity, and advanced mechanical proper- ties is under development. We cannot dismiss the effi- cacy and safety of new BVS technology. BVS by Abbott should focus on simple and short lesion. Various studies did not provide better outcome with long-term patency in tortuous, long, calcified lesions. Biotronik, biomime bioabsorbable stents came in and needs more compara- tive study. Consent for publication Consent for publication Not applicable. Not applicable. 13. Williams B, Mancia G, Spiering W, Agabiti Rosei E, Azizi M, Burnier M, Clement DL, Coca A, De Simone G, Dominiczak A, Kahan T (2018) 2018 ESC/ ESH Guidelines for the management of arterial hypertension: The Task Force for the management of arterial hypertension of the European Society of Cardiology (ESC) and the European Society of Hypertension (ESH). Eur Heart J 39(33):3021–3104 References 1. Giordano A, Ferraro P, Corcione N, Messina S, Maresca G, Coscioni E, Biondi-Zoccai G (2016) Successful treatment of recurrent carotid in-stent restenosis and drug-eluting balloon failure with a coronary bioresorbable vascular scaffold: a case report. Int J Surg Case Rep 21: 78–82. https://doi.org/10.1016/j.ijscr.2016.02.035 2. Chan W, Shah A, Džavík V, Overgaard CB (2014) Complex coronary artery bifurcation treatment utilizing everolimus-eluting bioresorbable vascular scaffolds and optical coherence tomography. Coron Artery Dis 25:629–631. https://doi.org/10.1097/MCA.0000000000000141 Authors’ contributions 7. Ielasi A, Tespili M (2014) Current and future perspectives on drug- eluting bioresorbable coronary scaffolds. Futur Cardiol 10:409–420. https://doi.org/10.2217/fca.14.14 ME put the concept and design of the work. AA collected data and acquisition analysis and was the major contributor in writing the manuscript. AS did the statistical analysis, drafted and revised the article. KS collected tables and figures, drafted and revised the article. IS analyzed and interpreted the patient data regarding BVS in Comparison to DES in Treatment of CTO and critical revision of article. All authors read and approved the final manuscript. 8. Kimura T, Kozuma K, Tanabe K, Nakamura S, Yamane M, Muramatsu T, Saito S, Yajima J, Hagiwara N, Mitsudo K, Popma JJ (2015) A randomized trial evaluating everolimus-eluting Absorb bioresorbable scaffolds vs. everolimus- eluting metallic stents in patients with coronary artery disease: ABSORB Japan. Eur Heart J 36:3332–3342. https://doi.org/10.1093/eurheartj/ehv435 9. Abizaid A, Costa RA, Schofer J, Ormiston J, Maeng M, Witzenbichler B, Botelho RV, Costa JR, Chamié D, Abizaid AS, Castro JP (2016) Serial multimodality imaging and 2-year clinical outcomes of the novel DESolve novolimus-eluting bioresorbable coronary scaffold system for the treatment of single de novo coronary lesions. JACC Cardiovasc Interv 9:565–574. https://doi.org/10.1016/j.jcin.2015.12.004 Availability of data and materials 10. Brugaletta S, Gomez-Lara J, Garcia-Garcia HM, Heo JH, Farooq V, van Geuns RJ, Chevalier B, Windecker S, McClean D, Thuesen L, Whitbourn R (2012) Analysis of 1 year virtual histology changes in coronary plaque located behind the struts of the everolimus eluting bioresorbable vascular scaffold. Int J Cardiovasc imaging 28:1307–1314. https://doi.org/10.1007/s10554-011-9981-4 Our prospective comparative cross-sectional study data used to support the findings of this study are available from the corresponding author upon request. Abbreviations h h 3. Dalos D, Gangl C, Roth C, Krenn L, Scherzer S, Vertesich M, Lang I, Maurer G, Neunteufl T, Berger R, Delle-Karth G (2016) Mechanical properties of the everolimus-eluting bioresorbable vascular scaffold compared to the metallic everolimus-eluting stent. BMC cardiovasc dis 16:104. https://doi.org/10.1186/s12872-016-0296-1 3. 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BMC cardiovasc dis 16:104. https://doi.org/10.1186/s12872-016-0296-1 2h-PPBG: 2-h post-prandial blood sugar; ABPM: Ambulatory blood pressure; AF: Atrial fibrillation; BVS: Bioresorbable vascular scaffold; CABG: Coronary artery bypass graft; CAD: Coronary artery disease; CRP: C-reactive protein; CTO: Chronic total occlusion; DES: Drug-eluting stents; ECG: Electrocardiogram; EDV: End-diastolic volume; EES: Everolimus-eluting stent; EF: LV ejection fraction; ESV: End-systolic volume; FBS: Fasting blood sugar; HBPM: Home blood pressure monitoring; HDL: High-density lipoprotein; IHD: Ischemic heart disease; LBBB: Left bundle branch block; LDL: Low-density lipoprotein; MACE: Major adverse cardiovascular events; MPI: Myocardial perfusion index; MSCT: Multi-slice CT coronary angiography; NHIS: National Health Interview Survey; OCT: Optical coherence tomography; OGTT: Oral glucose tolerance test; PCI: Percutaneous coronary intervention; QCA: Quantitative coronary angiography; RBBB: Right bundle branch block; TG: Triglycerides; TTE: Transthoracic echocardiography; TVR: Target vessel revascularization 2h-PPBG: 2-h post-prandial blood sugar; ABPM: Ambulatory blood pressure; AF: Atrial fibrillation; BVS: Bioresorbable vascular scaffold; CABG: Coronary artery bypass graft; CAD: Coronary artery disease; CRP: C-reactive protein; CTO: Chronic total occlusion; DES: Drug-eluting stents; 4. Dudek D, Onuma Y, Ormiston JA, Thuesen L, Miquel-Hebert K, Serruys PW (2012) Four-year clinical follow-up of the ABSORB everolimus- eluting bioresorbable vascular scaffold in patients with de novo coronary artery disease: the ABSORB trial. EuroIntervention 7:1060– 1061. https://doi.org/10.4244/EIJV7I9A168 5. Shehata IE, Cheng CI, Sung PH, Ammar AS, El-Sherbiny IA, Ghanem IG (2018) Predictors of myocardial functional recovery following successful reperfusion of acute ST elevation myocardial infarction. Echocardiography 35:1571–1578. https://doi.org/10.1111/echo.14106 5. Shehata IE, Cheng CI, Sung PH, Ammar AS, El-Sherbiny IA, Ghanem IG (2018) Predictors of myocardial functional recovery following successful reperfusion of acute ST elevation myocardial infarction. Echocardiography 35:1571–1578. https://doi.org/10.1111/echo.14106 6. 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https://openalex.org/W4292697579	https://indexlaw.org/index.php/conpedireview/article/download/3355/2876	Portuguese	null	Constitutionalism (s ) and reframing Hermeneutics : Reflections from the Critical Perspective Latin American	DOAJ (DOAJ: Directory of Open Access Journals)	2,016	cc-by	6,486	Resumo Em fins do século XX novos e difusos discursos do Direito vão apontando a emer- gência de modelos teóricos inovadores autodenominados “críticos”. No Brasil, neste contexto, se inaugura uma inovadora e revolucionária face do constitucionalismo que expande o poder do Judiciário, em nome da defesa da ordem democrática e direitos fun- damentais. Nesta esteira de mudanças uma das novidades é o amplo catálogo de direitos sociais que redefine o campo hermenêutico, até então herdeiro da tradição formal legalista moderna. Entretanto, no desejo de consolidar e garantir tanto no plano institucional como cultural os direitos fundamentais, se assiste na primeira década do século XXI no Brasil, bem como em alguns países latino americanos, a ampliação do campo da demo- cratização e políticas sociais. Neste marco, ao mesmo tempo que vai sendo implantado um novo paradigma constitucional a partir da plurinacionalidade, demodiversidade e novos direitos vinculados a uma nova racionalidade, há a expansão do papel do Poder Judiciário, o que acaba por povocar uma distorção da jurisdição e dos fundamentos her- menêuticos que lhe servem de legitimação. É a partir desta inédita complexidade que, desde o marco da tradição do pensamento crítico, pensadores do Direito são obrigados a repensar a hermenêutica a partir de uma nova lógica, novos fundamentos e novos ele- mentos epistemológicos, deslocando a questão hermenêutica para uma dimensão distinta do que lhe foi reservado pela racionalidade moderna e além do que até foi legado pela tradição positivista: o campo externo de valoração normativa. Desafio que entre oscilações e divergências não pode ser negado. Ivone Fernandes Morcilo Lixa Doutora em Direito Público pela Universidad Pablo de Olavide (UPO-UFSC) com pós doutoramento em Teoria do Direito (UFSC). Mestre em Direito pela Univer- sidade Federal de Santa Catarina. Professora da Universidade Regional de Blu- menau – FURB-SC do Curso de Direito. Pesquisadora e extensionista na área de Direitos Humanos, Hermenêutica Jurídica e Constitucionalismo Latino Americano Contemporâneo, com publicações nas referidas áreas. CONSTITUCIONALISMO(S) E RESIGNIFICAÇÃO HERMENÊUTICA: REFLEXÕES DESDE A PERSPECTIVA CRÍTICA LATINO AMERICANA Ivone Fernandes Morcilo Lixa Key words Contemporary constitutionalism; Legal hermeneutics; Critical theory; Fundamen- tal rights. Abstract At the end of XX century, news and diffuse law speeches will pointing the emergence of innovative theoretical models self-proclaimed “critical”. In Brazil, in this context, is inaugurated an innovative and revolutionary face of constitutionalism which expands the power of the judiciary, for the preservation of democratic order and fundamental rights. On this track changes a new feature is the extensive social rights catalog that redefines the hermeneutic field, hitherto of modern formal legalistic tradition. However, the desire to consolidate and ensure both institutional and cultural fundamental rights, is being wit- nessing the first decade of XX century in Brazil as well as in some Latin American coun- tries, expanding the field of democratization and social policies. In this context, while it is being implemented a new constitutional paradigm from plurinationality, demodiversity and new rights linked to rationality, there is the expansion of the role of the judiciary which ultimately provoque a distortion of jurisdiction and hermeneutic fundamentals that serves as its legitimacy. It is from this unprecedented complexity that since Mark of the tradition of critical thinking, the juridical thinkers are forced to rethink hermeneutics from a new logic, new foundations and new epistemological elements, shifting the her- meneutical question to a different dimension than have been given by modern rationality and beyond what was bequeathed to the positivist tradition: the external field of norma- tive valuation. Challenge between oscillations and divergences can not be overlooked. Palavras-chave Constitucionalismo contemporâneo; Hermenêutica jurídica; Teoria crítica; Direitos fundamentais. volume │ 01 245 e │ 01 01 iii encontro de internacionalização do conpedi – madrid iii encontro de internacionalização do conpedi – madrid a emergência de novos modelos. São tempos de “pós” e de novas políticas constitucionais que realinham as teorias e práticas jurídicas e é neste contexto que o saber subjugado e subalterno, o “colonial” ganha relevância. Desde uma perspectiva crítica e decolonial “interpretar” assume-se como atitude intelectual de reconhecimento da complexidade e da finalidade política do Direito, o que implica numa nova perspectiva hermenêutica que vai além da “questão metodológica”, deslocando a “questão” hermenêutica para a compreensão e reconhecimento do “plural”, superando-se as “verdades” univocistas do sentido do justo. 1. Introdução “Hermenêutica”, enquanto campo de saber específico relacionado como a “compre- ensão do sentido”, embora sendo uma inquietação intelectual constante ao longo da his- tória do pensamento ocidental desde a tradição grega, no contexto europeu moderno, ad- quire nova significação, reinventando-se como Teoria Geral de Interpretação sob o marco do paradigma colonizador moderno de ciência. Especificamente no campo do Direito, indo na mesma direção do modelo de racionalidade dominante, a hermenêutica jurídica, tendo como base teórica, ideológica e política o positivismo jurídico, se constrói como saber técnico instrumental cuja tarefa central foi a de estabelecer critérios tecnicamente válidos de interpretação exclusivamente do direito positivo, definindo-se, nesta perspec- tiva, como saber dogmático auxiliar e justificador da ciência jurídica. Em fins do século XX, os claros sinais de esgotamento do modelo paradigmático de ciência moderna e de projeto civilizatório, vão sendo construídos novos e difusos discursos que apontam para │ 01 volume 246 volume 2. Colocação do Problema: Hermenêutica e Direito (Paleopositivismos e Jusconstitucionalismos) A questão inicial que se coloca para qualquer campo do conhecimento é o seu pro- blema constitutivo. Ou seja, o desafio inicial do conhecimento é epistemológico, o que obriga delimitar e particularizar seu objeto de forma a evidenciar a problemática em torno da qual gravitam suas proposições, objetivos e finalidades tornando possível a identifi- cação de seus elementos constitutivos, e simultaneamente, definir seus paradigmas de sustentação e justificadores de sua legitimidade. Portanto, a primeira tarefa da reflexão aqui proposta é esclarecer o que se define como Hermenêutica, em particular a Hermenêutica Jurídica, e quais as questões essen- ciais que envolvem a delimitação de seu objeto e particularidades metodológicas, esforço que é um grande desafio aos que se dedicam a este campo do conhecimento que no Direi- to tem ocupado lugar privilegiado nestas primeiras décadas do século XXI. Sem dúvida uma das pautas centrais do Direito contemporâneo é a “questão herme- nêutica” e seus fundamentos legitimadores, sobretudo, quando as concepções ancoradas no velho paradigma juspositivista legalista ou paleo positivismo – definido pela intrínseca lógica auto sustentadora e auto justificadora legal - é substituído em meados da segunda década do século XX por um neojuspositivismo – subordinando o Direito ao sentido constitucional que, embora redefinido pelo princípio da legalidade substancial que vincula o sentido normativo à coerência dos princípios e fundamentos constitucionalmente esta- belecidos, mantém o velho paradigma da legalidade formal de produção –; e mais recen- temente, acentuadamente a partir das três últimas décadas do século XX e início do XXI, com a entrada em cena das democracias participativas os direitos fundamentais ganham novo status político e jurídico constituindo o que Ferrajoli chama da esfera do não decidível (FERRAJOLI, L. 2015, P.20), definindo limites políticos e jurídicos tanto da produção das normas como de sua interpretação. “Hermenêutica” é um termo herdado pela tradição do pensamento ocidental rela- cionado à compreensão do sentido para além do anunciado pela palavra, uma espécie de volume │ 01 247 │ 01 iii encontro de internacionalização do conpedi – madrid tarefa de transmutação compreensiva.1 Porém, o que atualmente se rotula como “Herme- nêutica” possui um sentido distinto daquele original e desenvolveu-se, em grande parte, sem ter consciência de si (GRONDIN, 1999, P. 22), como resultado de uma soma de condições sociais e teóricas ocorridas no cenário europeu moderno, e que acabou por construir-se como campo específico do conhecimento. 1 Embora sem possibilidade de esclarecimento conclusivo sobre a origem e o sentido da palavra hermèneutiké na cultura grega antiga, o certo é que era associada à atividade sacra, a transmutação do divino para o humano, e não reflexão sobre o sentido da linguagem tal qual passou a ser considerado na modernidade. 2. Colocação do Problema: Hermenêutica e Direito (Paleopositivismos e Jusconstitucionalismos) Por esta razão é questionável que se recue em períodos anteriores aos séculos XVI e XVII para ser escrita a historiografia do pensamento hermenêutico. Sob uma perspectiva histórica, a “questão hermenêutica” é colocada no mundo moderno nos campos do conhecimento relacionados com a interpretação de tex- tos, sobretudo com a teologia e jurisprudência, disciplinas teóricas que vão ser obrigadas a criar instrumentos técnicos segundo uma lógica cientifista acerca do conteúdo de textos transmitidos pela tradição através de uma correta interpretação de seu sentido. Sem querer mergulhar no imenso oceano da construção do pensamento hermenêu- tico moderno, o certo é que “Hermenêutica” ganha o status de Teoria Geral de Interpreta- ção como resultado de um processo cumulativo, possível pela conjunção, de um lado, do modelo de racionalidade científica moderna como “fiança” intelectual; e de outro, porque solucionou de maneira eficiente o conjunto de desafios colocados a partir do século XV no campo da interpretação dos textos sacros, profanos e jurídicos. Por esta razão lembra Gadamer que o sentido de “hermenêutica” é situada na tradição científica da moderni- dade. O uso moderno da palavra “hermenêutica” principia exatamente ao, quer dizer, com o surgimento do conceito moderno de método e de ciência. No seu uso aparece sempre implícita uma espécie de consciência metodológica. Não apenas possuímos a arte da interpretação como também podemos justificá-la teoricamente (GADAMER, 2004, P. 113). É exatamente neste sentido que vai se orienta o esforço teórico dos pensadores que delimitaram as propostas iniciais da moderna hermenêutica, qual seja, elaborar uma teoria a partir do conjunto das representações capaz de conferir auto-certeza científica ao conhecimento paulatinamente construído que atinge a “idade da Razão” no século XIX. Definida como saber específico acerca da compreensão do sentido “Hermenêutica” é um termo que historicamente foi carregado de imprecisões. Na tradição clássica, o grande esforço teórico em sua origem foi o de delimitar regras para eliminar as arbitrariedades e subjetivismos interpretativos, e com esta preocupação, é que se definiram as concepções univocistas, paradigmas dominantes entre os séculos XVII e XIX. Já no século XX amplia- se o campo metodológico e é reorientado seu objeto assumindo-se a Hermenêutica como filosofia universal da interpretação, considerando a interpretação a característica essencial 01 volume volume 248 volume iii encontro de internacionalização do conpedi – madrid da presença humana no mundo. Um avanço que acabou por definir o panorama do pen- samento hermenêutico contemporâneo. 2. Colocação do Problema: Hermenêutica e Direito (Paleopositivismos e Jusconstitucionalismos) Tanto a redução da realidade ao eterno relativo que condena o humano a impossibi- lidade do bem e do previsível como a incessante busca da verdade única e universal dada pelo experimental tecnicista podem ser considerados como extremos hermenêuticos que têm servido como mola propulsora ao que se pode definir como “pêndulo” hermenêu- tico2. Tal qual um pêndulo que oscila entre dois polos cuja velocidade diminui pouco a pouco, assim como a distância entre os extremos, mas sempre antagônicos por menor que seja a distância, até que chegue ao centro e pare; a Hermenêutica foi levada ao extremo univocista (que no Direito serve de instrumento operacional ao velho positivismo lega- lista) e seu contrário o relativismo equivocista (concepção que têm ocupado a discussão jurídica com a emergência do constitucionalismo contemporâneo). Evidentemente dois dogmas se excluem e simultaneamente reducionistas (DORANTES, 2005, P. 31). Segundo Mauricio Beuchot Puente (2005, Pgs. 46-47) a hermenêutica univocista sustenta a concepção reducionista de que somente há uma interpretação válida e todas as demais, em sua totalidade, são incorretas. Trata-se de uma construção que recebeu distintas matrizes vinculadas ao cientifismo que encontra seu apogeu no positivismo do século XIX e maior elaboração no positivismo lógico do século XX, segundo o qual, um enunciado era interpretado de maneira válida de acordo com o adequado procedimento lógico analítico de verificação independente da realidade, uma vez que a comprovação conceitual é tautológica. Além de que, lembra Beuchot, assim como afirmavam os es- colásticos que a universalidade constrói falsidades e a particularidade edifica a verdade é necessário ter cuidado com juízos extremos uma vez que é mais fácil haver equívoco no universal do que no particular. Ou seja, afirmar que apenas é corretamente interpretado o que é empiricamente verificável é uma afirmação em si mesmo insustentável já que a mente que enuncia o princípio é finita e assim o enunciado é em si mesmo inverificável. Ou seja, o univocismo se auto refuta. É exatamente este univocismo hermenêutico que vai ao encontro do modelo de ci- ência que vai presidir o Direito moderno (racional e universal) que se alia à consolidação das novas formas políticas resultantes da consolidação do Estado e provocam uma renova- ção no método hermenêutico na esfera jurídica. 2 A expressão “pêndulo” hermenêutico é utilizado por Arturo Guilhermo González Dorantes na obra Hacia una interpretación analógico-icónica del hombre (Coleção: Analogia Filosófica, nº 16, SIN 0188- 896X; México D.F., 2005) para designar as oscilações entre os extremos – do univocismo ao equivocismo – que tem conduzido as teorias hermenêuticas modernas. 2. Colocação do Problema: Hermenêutica e Direito (Paleopositivismos e Jusconstitucionalismos) Se um lado, o auge da filologia obriga um maior cuidado com o significado das palavras expressas nos textos e de outro, o desenvol- vimento de uma nova lógica jurídica que acredita ser possível descobrir o sentido objetivo volume │ 01 volume │ 249 01 iii encontro de internacionalização do conpedi – madrid da letra da lei no “sistema” normativo positivado conferem ao jurista os elementos para justificar e racionalizar sua tarefa: a de reconhecer e declarar o sentido objetivo da lei. No entender de Manuel Calvo García podem ser destacadas três consequências com o processo de estatização da lei: a) o direito deixa de ser aquele elemento neutro que se organizava a partir de uma necessidade interior, oculta, derivada de sua condição como verbum Dei ou de uma “razão” transformada em direito; b) se rompe com uma sociedade “juscêntrica” que fazia do descobrimento da lei pressuposto da organização do poder e se configurava aos juristas como estamento privilegiado na organização política da socie- dade medieval que estaria à mercê de sua intervenção tanto no descobrimento como na aplicação da lei; c) começam a serem definidos os pressupostos do positivismo jurídico. Portanto, o “direito criado” enfrenta os pressupostos de uma autoridade legisladora sedi- mentada nas manifestações dos doutos juristas (1994, P.38). Dessa maneira, na medida em que a autoridade quase “mágica” dos juristas vai per- dendo terreno frente às novas formas de exercício de poder do Estado, o direito deixa de ser um “direito de especialistas” no sentido estrito. A legitimidade hermenêutica da glosa e do comentário sofria então uma dupla perda: a da origem do texto legal e da negação da obscuridade de seu significado, já que até então, a autoridade quase divina de um texto legal e a opinião dos doutores garantia as exigências de segurança e certeza na fixação de um sentido objetivo da lei. Desde então, é necessário fundar uma nova legitimidade ao “velho” direito. Progressivamente o poder político é secularizado e se positiva o “velho” di- reito e as novas estruturas de poder culmina com um complexo processo de sistematização do direito que o coloca como objeto científico, como algo autônomo, frente ao intérprete. Na leitura de Lenio L. Streck o modus interpretativo vigente/dominante no cotidia- no dos juristas sustenta-se, paradoxalmente, em concepções herdadas exatamente desta velha na tradição positivista e na busca de sua superação. iii encontro de internacionalização do conpedi – madrid iii encontro de internacionalização do conpedi – madrid metade do século XX, quando o poder regulatório do Estado e a falência dos modelos sintáticos-semânticos de interpretação trazem para o primeiro plano o problema da in- determinação do sentido do Direito, encontrando em Kelsen uma forma de solução dos “desvios” subjetivistas criados pelas correntes hermenêuticas filiadas as Escolas do Direito Livre e da Jurisprudência dos Interesses. Desde então, superado o positivismo jurídico exegético e firmado o normativista, criaram-se teses e fundamentos hermenêuticos que entre discussões e aporias (tipo: volun- tas legis versus voluntas legislatoris ou objetivismos versus subjetivismos) acabam por situar a questão hermenêutica no campo metodológico, que mais servem como justificativa (álibis teóricos) de legitimação para os resultados que o intérprete se propõe a alcançar ou/e garantir, certezas que serão abaladas com o “giro” constitucionalista provocado pela emergência dos Estados Democráticos de Direito da segunda metade do século XX. 2. Colocação do Problema: Hermenêutica e Direito (Paleopositivismos e Jusconstitucionalismos) Em um primeiro momento – chamada pelo autor de positivismo primevo – é resolvido o problema da interpretação do direito sob uma perspectiva sintática. Neste caso, a simples determinação rigorosa da conexão lógica dos signos que compõe a “obra sagrada” (Código) seria o suficiente para resolver o problema da interpretação do direito. Assim, conceitos como o de analogia e princípios gerais do direito devem ser encarados também nessa perspectiva de construção de um quadro conceitual rigoroso que representaria as hipóteses – extrema- mente excepcionais – de inadequação dos casos às hipóteses legislativas (2011, P. 120). Entretanto, em um segundo momento, com a entrada do pensamento kelseniano é superada esta concepção. Define-se, no entender de Streck (2011, Pgs.120-121), um positivismo normativista, que surge a partir de propostas que aperfeiçoam o “rigor” lógico do cientificismo positivista e assim, ocorre uma modificação significativa já na primeira │ 01 volume volume 250 3. Constitucionalismo, Crítica e Resignificação Hermenêutica As últimas décadas do século XX são marcadas no plano teórico pelo esvaziamento das imagens e discursos representativos da racionalidade moderna, o que acaba por criar um complexo debate no qual são criadas novas rotulações. Instala-se um tempo dos “pós”, “de(s)” e “neo(s)”. Inéditas expressões que significam tentativas de rotular situações às quais ou se defende, e se tenta promover, ou se rechaça. Mas, há o que parece ser um ponto de convergência: o esgotamento das categorias da modernidade e das grandes uto- pias que serviram para construir o horizonte de futuro moderno, tomando-se a crítica à modernidade o ponto de partida para sua própria superação. Para autores como Slavoj Zizek (2012) a complexidade sem fim do mundo contem- porâneo possibilita o surgimento de conceitos opostos que parecem inquestionáveis, tais como a intolerância como tolerância, religião como senso comum racional, etc. Enfim vive-se um tempo em que é grande a tentação de gritar: “chega de bobagem”! Talvez seja essa, diz Zizek, a manifestação do desejo de estabelecer uma linha demarcatória entre a fala lúcida e sã e a bobagem, reação que tem servido para despertar a ira da ideologia predominante. Segue afirmando: O senso comum de nossa época diz que, em relação à antiga distinção entre “doxa”(opinião acidental/empírica, sabedoria) e Verdade, ou ainda mais ra- dicalmente, entre conhecimento positivo empírico e Fé absoluta, hoje é preciso traçar uma linha entre o que se pode pensar e o que se pode fazer (ZIZEK, 2012, p.20) É na tentativa de ir além do mero pensar, neste contexto dos “pós”/”de(s)”/ “neo(s)” e com desejo de fazer a reinvenção é que no Brasil se edificam e consolidam correntes no volume │ 01 lume │ 01 251 iii encontro de internacionalização do conpedi – madrid Direito que se auto denominam críticas. Em meados da década de 80 a realidade brasilei- ra reclamava a reconstrução da ordem democrática. Os instrumentos de exercício de po- der esvaziados pelo fim das verdades racionais que sustentaram durante séculos as formas de saber e de racionalidade dominantes não conseguiam mais responder inteiramente às inquietações e às necessidades daquele momento (WOLKMER,2012, P. 25) produzin- do, o que se passou a designar como “crise do direito”. A “crise”, produto da descrença e insegurança jurídica, é definida por Antonio Carlos Wolkmer como a agudização das contradições e dos conflitos sociais em dado processo histórico. 3 São inúmeros os pensadores do direito relacionados ao pensamento jurídico crítico, mas a verdadeira arqueologia epistemológica feita por Antonio Carlos Wolkmer na obra Introdução ao pensamento jurídico crítico, já citada, lembra com acuidade nomes e trajetórias que merecem ser registradas. 3. Constitucionalismo, Crítica e Resignificação Hermenêutica Expressa sempre a disfun- cionalidade, a falta de eficácia ou o esgotamento do modelo ou situação histórica aceitos e tradicionalmente vigentes (WOLKMER,2012, P.31). Sinais de esgotamento que vão conduzindo para o interior do campo jurídico o pensamento crítico, inaugurando, assim, uma discussão inédita e fértil. Uma possibilidade de enfrentamento e compreensão da “crise” jurídica brasileira foi encontrada na Teoria Crítica, concepção que desde a década de 60 vinha influenciado pensadores do direito europeu, através de idéias provindas do economicismo jurídico so- viético (Stucka, Pashukanis), da releitura gramsciana da teoria marxista feita pelo grupo de Althusser, da teoria crítica frankfurtiana e das teses arqueológicas de Foucault sobre o poder (WOLKMER, 2012, P.40). Na Europa as inovações da Teoria Crítica encontravam um terreno fértil no ambiente pós-guerra que projetavam no campo jurídico a desmistifica- ção do jusnaturalismo e do positivismo. Wolkmer retomando a trajetória do criticismo lembra que a crítica jurídica consolidou-se inicialmente na França por volta dos anos 70 culminando com o “manifesto” da Associação Crítica do Direito em 1978, atingindo em seguida a Itália, Espanha, Bélgica, Alemanha, Inglaterra e Portugal (WOLKMER, 2012, P.40). Na América Latina os “ventos” inovadores chegam por volta da década de 80 com o engajamento de juristas progressistas e comprometidos com a superação dos obstáculos políticos que impediam a construção e efetivação da democracia. Este movimento de renovação do pensamento jurídico recebe a adesão de pensadores brasileiros em inúme- ras faculdades de direito que acabaram por serem pioneiros de uma pedagogia jurídica emancipadora. As perspectivas epistemológicas, apesar de múltiplas, tinham como ponto em comum a defesa do rompimento com o positivismo legalista e revelação do caráter dominador e centralizador do direito hegemônico3. A Teoria Crítica trouxe consigo o impacto do questionamento do papel ideológico do direito na medida em que, diferentemente da concepção moderna de ciência, coloca no 252 01 iii encontro de internacionalização do conpedi – madrid interior da discussão jurídica as contradições e ambiguidades inerentes ao direito moder- no, buscando tomar o direito como instrumento não de manutenção da ordem estabele- cida, mas a possibilidade de emancipação do sujeito histórico tradicionalmente submerso em determinada normatividade repressora, mas também discutir e redefinir o processo de cons- tituição do discurso legal mitificado e dominante (WOLKMER, 2012, P.18). Mostrava-se assim um horizonte inovador, mas que trazia em si, a necessidade de rompimentos e abandonos teóricos. 4 Destacando-se a obra Direito Constitucional e a Efetividade das Normas, de Luis Roberto Barroso publicada no início da década de 90 e A Teoria Constitucional e o Direito Alternativo: para uma dogmática constitucional emancipatória In: Uma vida dedicada ao Direito: uma homenagem a Carlos Henrique de Carvalho publicada em 95. dogmática, tendo como eixo a concretização da Constituição (SARMEN- TO, 2010, P. 248). Desde aí se aprofundaram e se radicalizaram os estudos da hermenêutica jurídica. Influenciados pelo “giro” linguístico da filosofia e a entrada do pensamento de Ronald Dworkin, Robert Alexy, John Rawls, Hans Georg Gadamer, Jurgen Habermas, entre ou- tros, juristas como Lenio L. Streck e Eros Roberto Grau refundam o pensamento jurídico brasileiro denunciando e renunciando ao velho positivismo e seus procedimentos herme- nêuticos. Nesta nova etapa, ou quadra da história, como prefere Lenio L. Streck nominar este inédito momento de redefinição, é acentuada a natureza valorativa do Direito e dos princípios constitucionais. Nesse contexto, lembra Daniel Sarmento, há uma verdadeira febre de trabalhos sobre teoria dos princípios, ponderação de interesses, teorias da argumentação, proporcionalidade, razoabilidade, etc. (SARMENTO, 2010, P. 249) e se incorpora no pensamento jurídico crítico brasileiro o neoconstitucionalismo. Tratava-se de um momento de conquistas e necessidade de que fossem garantidas. Entretanto, já na primeira década do século XXI muitos se davam conta que o ne- oconstitucionalismo não era a superação do velho positivismo. Como afirma o próprio Lenio L. Streck, não é porque o neoconstitucionalismo tem um discurso axiologista e va- lorativo que é superado o positivismo formal legalista (STRECK, 2012, P. 37). As teorias “pós”/ “neo” positivistas acabaram por caírem na incerteza e indeterminação do Direito. Seguramente o “relativismo” e a “teoria da argumentação” foram mal incorporados no pensamento brasileiro e decreta-se a “morte do método” e em seu lugar passa a reinar ab- soluta incerteza e relativismo nas decisões judiciais. Possivelmente são os efeitos perversos de uma lógica colonizada que insiste em ser mantida na cultura jurídica brasileira. Não é novidade, desde Kelsen, que o julgador tem um “espaço discricionário” em aberto e que desde muito foi desmistificado o juiz “boca da lei”, mas como adverte Streck, deve-se estar atento com o “pós positivismo à brasileira”: ...é preciso estar alerta para certas posturas típicas do “pós positivismo à bra- sileira”, que pretende colocar o rótulo de novo em questões velhas, já bastan- te desgastadas nessa quadra da história, quando vivenciamos um tempo de constitucionalismo democrático. Ainda hoje presenciamos defesas vibrantes de “ativismos judiciais” para “implementar” e “concretizar” os direitos fun- damentais, tudo isso sempre retornando ao mesmo ponto: a ideia de que, no momento da decisão, o juiz tem um espaço discricionário no qual pode moldar sua “vontade”... (STRECK, 2012, P. 38). 3. Constitucionalismo, Crítica e Resignificação Hermenêutica Foi exatamente neste contexto que a hermenêutica ganha um novo status na discus- são jurídica, já que é próprio de momentos agudos de transição que a questão hermenêu- tica ganha relevância. Mais do que nunca foi necessário compreender a partir de novas categorias uma realidade também inovadora. A complexidade desta nova problemática de descobrir o “lugar” da hermenêutica numa lógica jurídica emancipadora fez com que fosse instaurada uma discussão movendo o “pêndulo hermenêutico”. A ordem política e jurídica colocada em marcha no Brasil com a Constituição de 1988 e o inédito momento histórico de então somados representavam a superação do au- toritarismo, exclusão social e violação de direitos fundamentais que, desde os primórdios da “invenção” colonialista, vinham constituindo uma patologia crônica exposta no grave quadro social que se delineava. Naquele momento, lembra Daniel Sarmento (2010), que grande maioria dos juristas entram em sintonia com as tendências constitucionalistas que apontavam como grande desafio garantir a efetividade das constituições democráticas. Até então, historicamente, os comandos jurídicos e políticos constitucionais, de fato, estavam nas mãos dos detentores dos poderes político, econômico e social e finalmente, o país co- meçou a “levar à sério” a Constituição e, apesar das dificuldades enfrentadas, tais como a desigualdade e o patrimonialismo que ainda povoam as instituições nacionais, os avanços em relação ao passado são inquestionáveis (SARMENTO, 2010, Pgs. 3-4). Logo após a homologação da Constituição de 1988 juristas como Luis Roberto Bar- roso e Clèmerson Merlin Clève4 passaram a militar a concepção de que a Constituição, enquanto norma jurídica deveria ser aplicada comumente pelos juízes, defendendo um “constitucionalismo de efetividade”, independente de qualquer mediação legislativa. ...o que viria a tirar do papel as proclamações generosas de direitos contidas na Carta de 88, promovendo justiça, igualdade e liberdade. Se até então, o discurso da esquerda era de desconstrução da dogmática jurídica, a doutrina da efetividade vai defender a possibilidade de um uso emancipatório da volume │ 01 253 01 iii encontro de internacionalização do conpedi – madrid dogmática, tendo como eixo a concretização da Constituição (SARMEN- TO, 2010, P. 248). iii encontro de internacionalização do conpedi – madrid iii encontro de internacionalização do conpedi – madrid método” o que acabou por produzir descontrolado soliptismo e ativismo judicial que mais confunde discricionariedade com arbitrariedade e o resultado, ao que parece, é que o “pêndulo hermenêutico” está “solto” e querendo voltar a um ponto “seguro”. Outra face perversa inegável produzida desde então é a crescente judicialização da vida cotidiana e política, o que acaba por neutralizar movimentos sociais e desvincular as práticas judiciais e políticas do Estado do compromisso constitucional primeiro, o que tem obrigado pen- sadores da Hermenêutica a rever fundamentos e pré concepções. Para L. Ferrajoli nestes tempos de total impotência e decadência da política predo- mina um constitucionalismo principialista, momento quando se leva em contra todas as suas implicações, coloca em perigo a separação dos poderes, o princípio da legalidade e sub- missão do juiz somente à lei: em síntese, todos os princípios do estado de direito (2012, Pgs. 245). Diante de angustiante constatação pergunta o pensador garantista italiano: quais alternativas se pode contrapor a esta orientação que coloca no Direito em uma espécie de “loteria do protagonismo judicial”? É momento de profundo e profícuo debate das vias possíveis de solução. Uma das possibilidades é apontada por L. Ferrajoli, ao propor como ponto de par- tida para a definição do horizonte hermenêutico os direitos fundamentais consagrados na ordem constitucional. ....isto é, não dar lugar a antinomia e lacunas, com todos os espaços de dis- cricionariedade política deixados em aberto, de um lado, pela proibição de produzir normas incompatíveis com os princípios constitucionais e, de outro, pelas possíveis formas e graus de observância da obrigação de sua atuação (2012, P.251). ....isto é, não dar lugar a antinomia e lacunas, com todos os espaços de dis- cricionariedade política deixados em aberto, de um lado, pela proibição de produzir normas incompatíveis com os princípios constitucionais e, de outro, pelas possíveis formas e graus de observância da obrigação de sua atuação (2012, P.251). Entretanto, chama a atenção L. Ferrajoli que todas as soluções, principalmente as mais controversas, não podem ser consideradas “verdadeiras” ou “objetivamente corretas”, uma vez que cada decisão, no campo hermenêutico, poderia ser considerada como condi- ções de possibilidade de decisão definidas a partir do horizonte compreensivo e, portanto, é inevitavelmente orientada por opções morais e políticas do intérprete (2012, P. 253). dogmática, tendo como eixo a concretização da Constituição (SARMEN- TO, 2010, P. 248). Em síntese, as concepções e modelos “descobertos” no Brasil em fins do século XX, sobretudo com a entrada em cena do neoconstitucionalismo, é decretada a “morte do │ 01 volume 254 5 Esta é a proposta defendida, entre outros, por Hélio Gallardo em Derechos Humanos como Movimiento Social. Edicioness desde abajo, Bogotá e explorada por Norman J. Solórzano Alfaro em Fragmentos de uma Reflexión Compleja sobre una Fundamentación Del Derecho y la Apertura a una Sensibilidad de Derecho Humano Alternativa, a ser publicado na Revista Jurídica Eletrônica nº 2 do Curso de Direito da Universidade Regional de Blumenau. iii encontro de internacionalização do conpedi – madrid E conclui o referido autor: Mas os juízes não serão nunca, porque não poderão nunca sê-lo, simples bo- cas da lei, como desejavam os iluministas. Nem poderão jamais alcançar verdades absolutas, mesmo que seja na forma da “verdadeira” resposta cor- reta. O reconhecimento desta imperfeição, ou se quiser, aporia, repito, é um fato de saúde institucional: gera o hábito da dúvida, a consciência do erro sempre possível, a disponibilidade para escutar todas as razões opostas que se confrontam no juízo, a “prudência” – a partir da qual advém o belo nome “ juris-prudência” – como estilo moral e intelectual da prática jurídica e, em geral, das nossas disciplinas (2012, P.254). volume │ 01 volume │ 01 volume │ 01 255 iii encontro de internacionalização do conpedi – madrid iii encontro de internacionalização do conpedi – madrid Em síntese, frente a complexidade do fenômeno jurídico contemporâneo e a perma- nente reconstrução e vigilância da ordem democrática no Brasil são possíveis múltiplas possibilidades de soluções para a “questão hermenêutica” uma vez que o legislador e nem mesmo o Estado são detentores de todas as hipóteses de interpretação e aplicação da nor- ma jurídica, o que evidentemente descortina a grande falácia do mito fundador do direito moderno: a certeza e segurança nascida da plena razão estatal. Portanto, em que pese o esforço de correntes hermenêuticas jurídicas contempo- râneas que se autoreferem como críticas, resta em aberto um espaço jurídico que inda não pôde ser preenchido pelas práticas fundadas nestas correntes. É possível pensar uma alternativa às práticas alternativas e reinventar a crítica desde as experiências democráticas participativas. Desde uma crítica à razão proléptica hermenêutica do direito moderno que além de contrair o presente reconhecendo como única fonte compreensiva o direito esta- tal, reduz o espaço de mediação jurídica ao Estado, é possível ampliar espaços presentes emergentes. Adotando a sugestão de Boaventura de Sousa Santos no que chama de sociologia das emergências que é a prática de ampliar o presente reconhecendo o que foi subtraído pela sociologia das ausências, hermeneuticamente ampliando os espaços de possibilidades de compreensão do direito para além do Estado, é possível identificar agentes, práticas e saberes com tendências de futuro sobre as quais é possível ampliar as expectativas de esperança. Trata-se de uma ampliação sobre as potencialidades e capacidades ainda não reconhecidas e necessariamente movendo-se no campo das experiências sociais que desde as práticas do “reconhecimento”, “transferência de poder” e “mediação jurídica” são legí- timos espaços de luta por dignidade humana e direitos fundamentais.5 É indo nesta direção que é possível falar-se em reconhecer o mundo social como mundo de possibilidade compreensiva e, portanto, fonte de uma nova racionalidade her- menêutica. Trata-se de uma perspectiva pluralista de direito que reconhece múltiplos es- paços de fontes normativas, apesar de na maioria das vezes, como lembra Antonio Carlos Wolkmer (1994, P.155) é informal e difusa. O pluralismo é uma fonte de inúmeras pos- sibilidades de regulação. Para Antonio Carlos Wolkmer O pluralismo enquanto concepção “filosófica” se opõe ao unitarismo determi- nista do materialismo e do idealismo modernos,pois advoga a independência e a inter-relação entre realidades e princípios diversos. iii encontro de internacionalização do conpedi – madrid naturais e cosmológicos, mas, igualmente, as condições de historicidade que cercam a vida humana. A compreensão filosófica do pluralismo reconhece que a vida humana é constituída por seres, objetos, valores, verdades, interesses e aspirações marcadas pela essência da diversidade, fragmentação, circunstan- cialidade, temporalidade, fluidez e conflituosidade. O pluralismo, enquanto “multiplicidade dos possíveis”, provém não só da extensão dos conteúdos ideológicos, dos horizontes sociais e econômicos, mas, sobretudo, das situações de vida e da diversidade das culturas. (1999,P.158) Em meio a discussão plural e decolonial nas primeiras décadas do século XXI che- gam ao poder em vários países latino americanos governos progressistas que avançaram no campo da democratização, políticas sociais e integração regional. Neste marco, os governos populares da Bolívia, Equador e Venezuela em especial, foram implantando um novo paradigma constitucional a partir da plurinacionalidade, demodiversidade, novos direitos vinculados a uma racionalidade reprodutiva da vida que expressamente deseja a vontade descolonizadora como conteúdo fundamental do projeto político em marcha nestas nações (MEDICI, 2012, P.56). Neste novo cenário, o processo hermenêutico jurídico não pode ser uma “canibaliza- ção”, para usar a expressão de Boaventura de Sousa Santos, dos demais. É necessário uma tradução das múltiplas hermenêuticas dentre as quais jurídica. E é neste sentido que não cabe uma hermenêutica jurídica nos moldes tradicionais. São campos distintos que se to- cam – o estatal e o social; o interno e o externo; o formal e o substancial – em que mundos normativos, práticas e saberes dialogam, se desentendem e interagem tornando possível reconhecer os pontos de contato entre a tradição moderna ocidental e os saberes leigos. A tarefa hermenêutica como tradução retoma o sentido mais original do termo,mas a partir de uma perspectiva inovadora que traduz saberes nem sempre convergentes. Com estas concepções o espaço hermenêutico no direito adquire uma dimensão dis- tinta do que tradicionalmente lhe foi reservado e vai um pouco mais além do que até foi edificado pela hermenêutica jurídica crítica dominante. É um espaço de aproximação e de assumir responsabilidades mútuas que rompe com a lógica construída pelo saber coloni- zador e abre para ainda tornar possível a esperança no justo. As condições de possibilidade de compreensão é elaborada com o outro e a partir deste outro historicamente negado e silenciado, reinventando processos hermenêuticos desde uma ordem constitucional de- mocrática e participativa. iii encontro de internacionalização do conpedi – madrid Parte-se do princípio de que existem muitas fontes ou fatores causais para explicar não só os fenômenos 01 volume 256 volume iii encontro de internacionalização do conpedi – madrid 4. Conclusões A Hermenêutica Jurídica ao longo da modernidade define seu objeto e pressupostos teóricos dentro de preocupações que foram oscilando entre dois campos metodológicos volume │ 01 volume │ 01 257 lume │ 01 iii encontro de internacionalização do conpedi – madrid aparentemente excludentes: o univocismo, de matriz iluminista que no Direito alimentou e justificou o legalismo formal positivista; e o relativismo de matriz romancista que mais recentemente foi incorporado equivocadamente pelo neoconstitucionalismo panprinci- pialista – como chamam seus críticos; o que tem delineado uma insistente atitude cientifi- cista “pendular”. Tal discussão, incorporada no pensamento jurídico moderno, foi repro- duzida pela cultura jurídica brasileira desde o século XIX, inicialmente com forte matriz univocista e que, no estágio contemporâneo do Direito, sobretudo com as chamadas Teorias Críticas que ganharam relevo após a instauração do Estado Democrático de Direi- to pós 1988, se desloca para o relativismo soliptista. A emergência do constitucionalismo contemporâneo pautado no horizonte da democracia substancial – ou participativa – que tem orientado os modelos políticos-jurídicos construídos em alguns países da América Latina nas últimas décadas do século XX refundam a lógica de Estado e de Direito. As- sim, são reinventadas práticas hermenêuticas com novas metodologias, pedagogicamente democráticas. O presente estudo pretendeu refazer brevemente a trajetória do pensamen- to hermenêutico jurídico moderno brasileiro destacando os desafios contemporâneos a serem superados buscando discutir o esgotamento dos modelos tradicionais e as novas possibilidades a partir da criticidade pautada em modelos políticos e jurídicos democrá- ticos participativos que têm redefinido o paradigma hermenêutico em direção inédita e desafiadora. 5. Referências BEUCHOT PUENTE, Mauricio. Tratado de Hermenêutica Analógica – hacia un nue- vo modelo de interpretación. 3ª Ed., Colección Seminarios. Editorial Ítaca: UNAM, México D.F., 2005. DORANTES, Arturo Guilhermo González, Hacia una interpretación analógico-icónica del hombre Coleção: Analogia Filosófica, nº 16, SIN 0188-896X; México D.F., 2005. FERRAJOLI, Luigi. A democracia através dos direitos – o constitucionalismo garantista como modelo teórico e como projeto político. Tradução de Alexandrer Araújo de Souza e ou- tros. São Paulo: Ed. RT, 2015. GADAMER, Hans-Georg. Verdade e Método II – Complementos e Índice. Tradução de Enio Paulo Giachini. São Paulo: Vozes, 2004. GARCÍA, Manuel Calvo. Los fundamentos del método jurídico: una revisión crítica. Ma- drid: Tecnos, 1994. GRONDIN, Jean. Introdução à hermenêutica filosófica. Tradução de Benno Dischinger. São Leopoldo: Ed. Unisinos, 1999. MÉDICI, Alejandro. La constitucionalización horizontal – teoria constitucional y giro deco- lonial.San Luis de Potosí: Universidad Autónoma de San Luis Potosí, 2012. 258 │ 01 iii encontro de internacionalização do conpedi – madrid iii encontro de internacionalização do conpedi – madrid SARMENTO, Daniel. Por um constitucionalismo inclusivo: história constitucional brasilei- ra, teoria da constituição e direitos fundamentais. Rio de Janeiro: Lumen Juris, 2010. STRECK, Lenio L. Hermenêutica Jurídica e(m) Crise – uma exploração hermenêutica da construção do Direito. 10ª Ed., Porto Alegre: Livraria do Advogado, 2011. WOLKMER, Antonio Carlos. Introdução ao Pensamento Jurídico Crítico. 8ª ed. São Pau- lo: Saraiva, 2012 ______. Pluralismo Jurídico – fundamento de uma nova cultura no Direito. São Paulo: Editora Alfa Omega, 1994. ZIZEK, Slavoj. Em defesa das causas perdidas. Tradução de Maria Beatriz de Medina. São Paulo: Bointempo, 2012. volume │ 01 volume │ 01 volume │ 01 259 259
https://openalex.org/W4390709120	https://periodicorease.pro.br/rease/article/download/12789/6053	Portuguese	null	ABORDAGENS MULTIDISCIPLINARES NO TRATAMENTO DE DOENÇAS CRÔNICAS	Revista Ibero-Americana de Humanidades, Ciências e Educação	2,024	cc-by	3,207	Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE doi.org/10.51891/rease.v9i12.12789 doi.org/10.51891/rease.v9i12.12789 1 Faculdade Pitágoras de Feira de Santana. 2 Faculdade Venda Nova do Imigrante. 3Universidade Tiradentes. 4Universidade Salvador. 5 Faculdade Anísio Teixeira. 6 UNIFTC. 7Faculdade Anísio Teixeira. 8Faculdade Pitágoras de Feira de Santana. 9Universidade Federal do Maranhão. 10 Unifacs. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 1 Faculdade Pitágoras de Feira de Santana. Keywords: Multidisciplinary Approaches. Chronic Diseases. Interdisciplinary Collaboration. ABORDAGENS MULTIDISCIPLINARES NO TRATAMENTO DE DOENÇAS CRÔNICAS Nayara Tharcylla dos Santos Silva Bomﬁm1 Stephanie Tolêdo Santiago2 Alice de Jesus Santos3 Fhadilla de Oliveira Santos4 Elielza Costa Araújo5 Erick Vanderley Gondim Lopes6 Alana Matos Biao7 Thaís de Jesus Santos Silva8 Thais Regina Ferreira França9 Caroline Brandão Brito10 RESUMO: Esta revisão bibliográfica científica analisa a eficácia das abordagens multidisciplinares no tratamento de doenças crônicas, destacando a importância da colaboração entre profissionais de saúde de diversas disciplinas. A síntese das evidências revela benefícios consistentes na melhoria dos desfechos clínicos e na promoção de uma abordagem holística à saúde. Desafios, como a coordenação interdisciplinar e a equidade no acesso, são discutidos, enfatizando a necessidade de políticas e práticas que apoiem a implementação eficaz dessas estratégias inovadoras. As considerações finais apontam para a personalização do tratamento, a necessidade de estudos de longo prazo e a importância de direcionar pesquisas futuras para otimizar a integração interdisciplinar na prática clínica. 395 Palavras-chave: Abordagens Multidisciplinares. Doenças Crônicas. Colaboração Interdisciplinar. Palavras-chave: Abordagens Multidisciplinares. Doenças Crônicas. Colaboração Interdisciplinar. ABSTRACT: This scientific literature review examines the effectiveness of multidisciplinary approaches in the treatment of chronic diseases, emphasizing the importance of collaboration among healthcare professionals from diverse disciplines. The synthesis of evidence reveals consistent benefits in improving clinical outcomes and promoting a holistic approach to health. Challenges, such as interdisciplinary coordination and equity in access, are discussed, underscoring the need for policies and practices that support the effective implementation of these innovative strategies. The concluding remarks emphasize treatment personalization, the necessity for long-term studies, and the importance of directing future research to optimize interdisciplinary integration in clinical practice. Keywords: Multidisciplinary Approaches. Chronic Diseases. Interdisciplinary Collaboration. 1 Faculdade Pitágoras de Feira de Santana. 2 Faculdade Venda Nova do Imigrante. 3Universidade Tiradentes. 4Universidade Salvador. 5 Faculdade Anísio Teixeira. 6 UNIFTC. 7Faculdade Anísio Teixeira. 8Faculdade Pitágoras de Feira de Santana. 9Universidade Federal do Maranhão. 10 Unifacs. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE RESUMEN: Esta revisión bibliográfica científica examina la eficacia de los enfoques multidisciplinarios en el tratamiento de enfermedades crónicas, destacando la importancia de la colaboración entre profesionales de la salud de diversas disciplinas. La síntesis de evidencias revela beneficios consistentes en la mejora de los resultados clínicos y la promoción de un enfoque holístico de la salud. Se abordan desafíos, como la coordinación interdisciplinaria y la equidad en el acceso, enfatizando la necesidad de políticas y prácticas que respalden la implementación efectiva de estas estrategias innovadoras. Las consideraciones finales señalan hacia la personalización del tratamiento, la necesidad de estudios a largo plazo y la importancia de dirigir futuras investigaciones para optimizar la integración interdisciplinaria en la práctica clínica. Palabras clave: Enfoques Multidisciplinarios. Enfermedades Crónicas. Colaboración Interdisciplinaria. INTRODUÇÃO As doenças crônicas representam um desafio significativo para os sistemas de saúde global, constituindo uma parcela substancial da carga de morbidade e mortalidade. Diante da complexidade inerente a condições como diabetes, hipertensão, doenças cardiovasculares e respiratórias crônicas, a busca por estratégias terapêuticas mais eficazes torna-se imperativa. A abordagem convencional, predominantemente centrada na intervenção médica isolada, tem mostrado limitações na gestão abrangente e sustentável dessas patologias. Assim, este trabalho explora a crescente importância das abordagens multidisciplinares no tratamento de doenças crônicas, reconhecendo a necessidade de integrar diversas disciplinas para proporcionar uma resposta abrangente e adaptativa às complexidades dessas condições de saúde. 396 A prevalência das doenças crônicas continua a aumentar em todo o mundo, sendo exacerbada por fatores como envelhecimento populacional, urbanização e mudanças nos estilos de vida. Este cenário demanda uma revisão crítica das estratégias terapêuticas convencionais, ressaltando a importância de uma visão mais holística. As abordagens multidisciplinares emergem como um paradigma promissor, abrangendo não apenas a intervenção médica, mas também a incorporação de profissionais de saúde de diversas disciplinas, como nutricionistas, psicólogos, fisioterapeutas e assistentes sociais. A compreensão crescente da interconexão entre fatores biológicos, psicossociais e ambientais nas doenças crônicas destaca a inadequação das abordagens unidimensionais. A influência mútua entre aspectos psicológicos, comportamentais e fisiológicos na progressão e manejo dessas condições destaca a necessidade de uma abordagem interdisciplinar que considere integralmente o paciente. Dessa forma, as estratégias terapêuticas devem Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE transcender os limites das disciplinas tradicionais, visando proporcionar um cuidado mais completo e personalizado. A literatura científica recente tem documentado o impacto positivo das abordagens multidisciplinares no tratamento de doenças crônicas. A integração de diferentes especialidades permite uma avaliação mais abrangente dos fatores de risco, uma personalização mais eficaz dos planos de tratamento e uma promoção mais eficiente da adesão do paciente. Este trabalho busca sintetizar e contextualizar as evidências disponíveis, destacando as contribuições específicas de cada disciplina na gestão global de doenças crônicas e fomentando uma compreensão mais aprofundada dos benefícios derivados dessa abordagem integrada. Em síntese, à medida que a prevalência de doenças crônicas continua a desafiar os sistemas de saúde, a implementação de abordagens multidisciplinares surge como uma estratégia inovadora e necessária. INTRODUÇÃO Este trabalho visa fornecer uma visão abrangente das bases teóricas e práticas que sustentam a eficácia dessas abordagens, destacando seu papel na promoção da saúde, prevenção de complicações e melhoria da qualidade de vida para aqueles afetados por doenças crônicas. 397 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN 2675 3375 METODOLOGIA A metodologia empregada nesta revisão bibliográfica tem como objetivo explorar e sintetizar a crescente evidência científica relacionada às abordagens multidisciplinares no tratamento de doenças crônicas. O processo metodológico adotado fundamenta-se em diretrizes reconhecidas para revisões bibliográficas sistemáticas, promovendo a identificação, seleção e análise crítica de estudos relevantes. A seguir, descreve-se de maneira detalhada as etapas seguidas durante a condução desta revisão. A definição de uma pergunta de pesquisa clara e específica é o ponto de partida. Neste caso, a pergunta focaliza a eficácia das abordagens multidisciplinares no tratamento de doenças crônicas, explorando os benefícios, desafios e impactos nos desfechos clínicos. Foram consultadas bases de dados científicas amplamente reconhecidas, incluindo PubMed, Scopus e Web of Science. A busca abrangeu artigos publicados até a data limite desta revisão, com termos de pesquisa abrangentes relacionados a "abordagens multidisciplinares" e "doenças crônicas". A seleção dos estudos baseou-se em critérios pré-definidos. Foram incluídos artigos que abordavam especificamente intervenções multidisciplinares no tratamento de doenças , ç ISSN - 2675 – 3375 ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE crônicas, englobando diferentes disciplinas de saúde. Estudos que não atendiam a esse critério ou que não apresentavam relevância para a temática foram excluídos. Os dados foram extraídos de forma sistemática dos artigos selecionados. Informações como características dos estudos, participantes, intervenções multidisciplinares empregadas e resultados clínicos foram registrados de maneira organizada para análise subsequente. A análise crítica dos estudos incluídos foi realizada, destacando resultados significativos, metodologias utilizadas e limitações identificadas. A síntese dos dados permitiu a construção de uma narrativa coesa, explorando os achados relevantes e evidenciando tendências ou lacunas no conhecimento atual. A qualidade metodológica dos estudos foi avaliada utilizando critérios específicos relacionados ao desenho do estudo, tamanho da amostra, métodos de intervenção e análise estatística. Esta avaliação contribuiu para contextualizar os resultados e oferecer uma visão crítica da robustez das evidências apresentadas. Os resultados foram apresentados de forma clara e concisa, seguindo a estrutura pré- definida. Destacaram-se os principais achados, tendências e implicações práticas, oferecendo uma visão abrangente do estado atual do conhecimento sobre abordagens multidisciplinares no tratamento de doenças crônicas. 398 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN 2675 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 RESULTADOS A análise sistemática dos estudos selecionados revelou uma convergência significativa de evidências que apoiam a eficácia das abordagens multidisciplinares no tratamento de doenças crônicas. A síntese abrangente dos dados indica que intervenções que incorporam profissionais de saúde de diversas disciplinas desempenham um papel crucial na gestão abrangente e adaptativa dessas condições de saúde complexas. Os resultados destacam a heterogeneidade nas intervenções multidisciplinares avaliadas, abrangendo desde programas de educação do paciente até intervenções terapêuticas intensivas. Uma constante, no entanto, foi a observação de melhorias significativas nos desfechos clínicos quando comparadas às abordagens unidimensionais convencionais. A inclusão de profissionais como nutricionistas, psicólogos, fisioterapeutas e assistentes sociais demonstrou influenciar positivamente a adesão ao tratamento, modificação de comportamento e gestão eficaz de fatores de risco associados a doenças crônicas. Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Além disso, os estudos revisados destacaram a relevância do aspecto psicossocial no tratamento das doenças crônicas. Intervenções que abordam não apenas os aspectos físicos, mas também os emocionais e sociais, demonstraram resultados mais expressivos na melhoria da qualidade de vida dos pacientes. A colaboração entre disciplinas mostrou-se crucial para a identificação e abordagem eficaz de barreiras emocionais, redução do estigma e promoção de estratégias de enfrentamento positivas. A revisão também evidenciou a necessidade de uma abordagem personalizada, considerando a heterogeneidade dos pacientes e a diversidade nas manifestações das doenças crônicas. Estratégias que incorporaram a individualização do tratamento, adaptando-se às necessidades específicas de cada paciente, demonstraram resultados mais expressivos em comparação com intervenções padronizadas. Contudo, alguns desafios emergiram nos resultados, incluindo a necessidade de uma colaboração interdisciplinar eficaz, a integração de sistemas de saúde e a garantia de uma comunicação fluida entre os profissionais envolvidos. Além disso, a escassez de estudos de longo prazo e a avaliação mais aprofundada dos custos associados a essas abordagens ressaltam lacunas significativas na literatura atual. 399 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN 2675 3375 DISCUSSÃO A síntese e análise dos resultados obtidos nesta revisão bibliográfica destacam a relevância e a eficácia das abordagens multidisciplinares no tratamento de doenças crônicas. A discussão dos principais achados destaca nuances importantes que informam a compreensão atual sobre o impacto dessas intervenções, bem como identifica áreas de oportunidade e desafios a serem abordados no contexto clínico e de pesquisa. Os resultados consistentemente positivos observados nas intervenções multidisciplinares corroboram a ideia de que a inclusão de profissionais de saúde de diversas disciplinas contribui para uma gestão mais completa e adaptativa das doenças crônicas. A abordagem holística, considerando não apenas os aspectos físicos, mas também os emocionais e sociais, parece ser crucial para melhorar a qualidade de vida dos pacientes e otimizar os resultados clínicos. A atenção ao paciente como um ser integral, em vez de focar exclusivamente na doença, emerge como uma abordagem paradigmática na promoção da saúde a longo prazo. A personalização do tratamento, adaptando as intervenções às necessidades individuais dos pacientes, é uma constatação crucial nesta discussão. A heterogeneidade nas ISSN - 2675 – 3375 ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE manifestações e nas respostas ao tratamento destaca a importância de estratégias flexíveis e adaptáveis. Essa abordagem personalizada não apenas otimiza os resultados clínicos, mas também contribui para uma maior adesão ao tratamento, combatendo a resistência e promovendo mudanças positivas no comportamento do paciente. Entretanto, os desafios também são evidentes. A colaboração interdisciplinar, embora fundamental, muitas vezes encontra obstáculos relacionados à comunicação e coordenação entre os profissionais de saúde de diferentes disciplinas. A integração eficaz dessas abordagens multidisciplinares nos sistemas de saúde, juntamente com a sustentabilidade financeira a longo prazo, emerge como questões críticas que necessitam de atenção. A escassez de estudos de longo prazo é outra limitação notável na literatura revisada. Embora os resultados positivos sejam encorajadores, a compreensão dos efeitos a longo prazo dessas abordagens multidisciplinares é essencial para avaliar a sustentabilidade e a durabilidade dos benefícios observados. Pesquisas futuras devem focar em estudos prospectivos de longo prazo para preencher essa lacuna de conhecimento. Além disso, é vital abordar as disparidades no acesso a intervenções multidisciplinares. A discussão sobre como tornar essas abordagens mais acessíveis e equitativas é crucial para garantir que todos os pacientes, independentemente de sua localização geográfica ou status socioeconômico, possam se beneficiar dessas estratégias inovadoras. DISCUSSÃO 400 Em resumo, as discussões baseadas nos resultados desta revisão destacam que as abordagens multidisciplinares no tratamento de doenças crônicas representam um avanço significativo na prática clínica. Embora os benefícios sejam evidentes, é imperativo superar desafios como a coordenação interdisciplinar, a integração nos sistemas de saúde e a consideração das disparidades de acesso para efetivamente incorporar essas estratégias na prestação de cuidados de saúde a longo prazo. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 CONSIDERAÇÕES FINAIS A robustez dos resultados obtidos nesta revisão evidencia a eficácia das abordagens multidisciplinares no tratamento de doenças crônicas. A inclusão de profissionais de diversas disciplinas não apenas melhora os desfechos clínicos, mas também promove uma visão mais holística da saúde, considerando os aspectos físicos, emocionais e sociais. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE A necessidade de personalização do tratamento, adaptando as intervenções às características individuais dos pacientes, emerge como uma consideração crucial. A heterogeneidade nas manifestações das doenças crônicas reforça a importância de estratégias flexíveis e adaptáveis para otimizar os resultados e promover uma maior adesão ao tratamento. Os desafios identificados, como a coordenação interdisciplinar e a integração nos sistemas de saúde, destacam áreas que exigem atenção contínua. Superar essas barreiras permitirá uma implementação mais eficaz das abordagens multidisciplinares na prática clínica. Além disso, a consideração das disparidades no acesso e a equidade na entrega dessas intervenções são essenciais para garantir que todos os pacientes possam se beneficiar dessas estratégias inovadoras. A lacuna na evidência relacionada aos efeitos a longo prazo das abordagens multidisciplinares destaca a necessidade premente de estudos prospectivos de longo prazo. Compreender a durabilidade dos benefícios observados e identificar estratégias sustentáveis é essencial para fundamentar a implementação generalizada dessas intervenções. As implicações práticas derivadas desta revisão indicam a necessidade de promover a integração interdisciplinar na prática clínica e nos sistemas de saúde. A incorporação de abordagens multidisciplinares deve ser apoiada por políticas que incentivem a colaboração, a formação interprofissional e a alocação adequada de recursos. 401 Como área em constante evolução, as pesquisas futuras devem se concentrar na ampliação do conhecimento sobre intervenções específicas, na identificação de modelos de colaboração eficazes e na exploração de estratégias inovadoras. A pesquisa translacional que traduz efetivamente a evidência científica para a prática clínica é essencial para maximizar o impacto das abordagens multidisciplinares. Em conclusão, esta revisão reforça a importância das abordagens multidisciplinares no tratamento de doenças crônicas. Ao considerar os desafios existentes, abordar disparidades e promover a colaboração interdisciplinar, é possível otimizar a prestação de cuidados, melhorar os resultados clínicos e aprimorar a qualidade de vida dos pacientes afetados por doenças crônicas. REFERÊNCIAS AMERICAN Diabetes Association. 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Effectiveness of chronic obstructive pulmonary disease-management programs: systematic review and meta-analysis. American Journal of Medicine, 124(11), 1056-e7. STARFIELD, B. (1998). Primary care: balancing health needs, services, and technology. Oxford University Press. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.12. dez. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE TSAI, A. C., Morton, S. C., Mangione, C. M., Keeler, E. B., Parkerton, P. H., & Kahn, K. L. (2005). A meta-analysis of interventions to improve care for chronic illnesses. American Journal of Managed Care, 11(8), 478-488. WAGNER, E. H. (1998). Chronic disease management: what will it take to improve care for chronic illness?. Effective clinical practice, 1(1), 2-4. WAGNER, E. H., & Bennett, S. M. (2010). Austin Bt, Greene SM, Schaefer JK, et al. Finding common ground: patient-centeredness and evidence-based chronic illness care. 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https://openalex.org/W4382762658	https://zenodo.org/record/5346311/files/Proceedings_Chalier_pdf.pdf	English	null	Aromatic profile of wheat flour and bran fractions	null	2,021	cc-by	2,784	E. Guichard & J.L. Le Quéré (Eds): Proc. 16th Weurman Flavour Research Symposium, 2021. DOI:10.5281/zenodo.5346311 Abstract The major output of milling process of wheat grains is flour, bran represents the most important fraction of all by-products. Its content in fibres and micro-nutrients makes its enrichment interesting in breads and cereals products if no unfavourable changes in the sensorial properties are induced. In this research, we proposed to compare the volatile components of flour, fine and coarse brans to evaluate the potential impact of addition of bran in flour. The three fractions were obtained by using a micro- mill and the aromatic profile was characterized by GC-MS after SPME extraction. Numerous compounds due to lipid oxidation such as hexanal, 1-octen-3ol, nonanal, 2-heptanone were found in the three fractions. Limonene, a terpenic compound, characterized by sweet, citrus and peely odour was identified in the three fractions. This unexpected compound was already identified in flour. Eugenol, a phenolic compound, with spicy, clove like, woody odour and phenolic savour was only identified in the brans fraction. This compound is formed via the general phenylpropanoid pathway, including the conversion of coniferyl alcohol and specific enzyme. It could provide a specific marker of bran addition in flour. Keywords: aroma profile, bran, flour, marker, SPME, wheat Aromatic profile of wheat flour and bran fractions José Daniel Wicochea Rodríguez1, Cécile Barron1, Valérie Lullien-Pellerin1, Peggy Rigou2 and PASCALE CHALIER1 1 IATE, Univ. Montpellier, INRAE, Institut Agro, Montpellier, France, pascale.chalier@umontpellier.fr 2 SPO, Univ. Montpellier, INRAE, Montpellier, France Texte 1 IATE, Univ. Montpellier, INRAE, Institut Agro, Montpellier, France, pascale.chalier@umontpellier.fr 2 SPO, Univ. Montpellier, INRAE, Montpellier, France Texte Introduction Wheat grain is an indehiscent dry fruit consisting of a single seed intimately welded to different envelopes or layers. The seed was constituted of the starchy endosperm and the aleurone layer, embryonic axis and the scutellum and the nucellar epidermis and the testa. Each of these tissues has a particular structure and composition [1]. The milling process has for aim to recover the major part of the starchy endosperm into flour, a rich source of starch and proteins that is used to make cereal-based foods products. The flour extraction rate varies classically between 70 and 77%. Other fractions obtained by milling are coarse, fine brans and shorts. Wheat bran is composed with the pericarp, testa, hyaline and aleurone layer but also contains part of the peripheral starchy endosperm. Bran represents the most important fraction of overall by-products accounting for around 15-25% of the grain weight. It contains dietary fibres (45-55%), proteins (13-18%), minerals (ash 3.4-8%), lipids (3-4%) and micro-nutrients as antioxidant component and therefore its addition could improve breads and cereals products nutritional values if potential contaminants (heavy metals, pesticides, mycotoxins) are carefully checked and conform to the prevailing legislation limits [2]. However, bran addition in the consumable products can also induce changes in the sensorial properties such as texture of the dough or undesirable taste and flavour. In this research, we proposed to compare the volatile components of flour, fine and coarse brans to evaluate the potential impact of addition of bran in flour. Indeed, if the volatile compounds of flour have already been identified, few data exist about the volatile compounds present in the brans fractions after milling. Proceedings of the 16th Weurman Flavour Research Symposium Materials ommon wheat (Triticum aestivum L.) grown in organic conditions was purchased from Salvagnac Agrib n (Salvagnac, France). P. Chalier et al. P. Chalier et al. P. Chalier et al. Extraction and analysis Headspace Solid Phase Micro-Extraction (HS-SPME) was used to analyse the different fractions obtained after milling. Samples of 0.5 g were deposited in 20 ml vial. For semi-quantification, 5 µl of internal standard solution was added (0.2 g/l of 2-heptanol in distilled water). Each sample was pre-incubated for 5 min at 50°C and extracted during 30 min at 50°C using fibre (30/50 µm DVB/CAR/PDMS, stableflex 2 cm 23 Ga).The desorption was carried out for 5 min at 250°C in the automatic Combipal injector. Between each measurement the fibre was baked-out for 30 min at 270°C. GC-MS ISQ (ThermoScientific, Austin, Texas, USA) equipped with a DB-WAX polar capillary column (30 m, 0.25 mm i.d. x 0.25 µm of thickness) and a quadrupole detector was used for identification. Helium was used as carrier gas with a flow rate of 1.2 ml/min. The oven temperature was kept at 40°C for 5 min and programmed to 250 °C at a rate of 2°C/min. Spectra were obtained in the electron impact mode with 70 eV. The full scan mode was used, and the range of scans was between 40-500 amu. Compounds were identified by using different libraries (INRA, NIST, Wiley) and linear retention indices calculation. Quantitatively, the flour was less rich in volatile compounds (around to 2mg/kg dry basis) than fine and coarse brans (around 8mg/kg dry basis). As derived product of lipid represented the major part of volatiles components, it can be related to the lowest lipid content of flour compared to fine and coarse brans, 1.6, 5.3 and 4.7 %, respectively. Milling The wheat grains were tempered to reach 16.5 % (w/w) of moisture content. A micro-mill was used to simulate the industrial milling process. The process of milling is divided in four steps including two breaking stages, one sizing and one reduction stage leading to 4 fractions; flour, fine bran, coarse bran and shorts. Each step consisted of a size reduction phase and a sieving phase. For the last two phases, the flour obtained after milling was processed with a bran finisher (CHOPIN S.A) for 1.5 min. The total flour obtained corresponds to an extraction rate higher than 70%. Aromatic profile of wheat flour and bran fractions It is important to highlight that the studied wheat was cultivated in organic conditions. Another hypothesis was the contamination by fungi able to produce terpene compounds. In flour, three other compounds previously detected [3], were formed by other ways that lipid oxidation. Acetic acid can be a sign of natural grain contamination by acetic bacteria or other microorganisms. Benzyl-alcohol could occur from phenylalanine via benzaldehyde either by β-oxidative pathway and non-oxidative way. This compound was present in flour and in fine bran but not in coarse bran indicating potential difference in enzymes equipment or protein amount (e.g., more germ in fine than in coarse). While the presence of 2-ethylhexanol has been already detected in flours, its origin requires further investigations. Indeed, it is known as an indoor air pollutant with human toxicity [4]. Its detection in flour only is surprising and could be problematic. In bran fractions, two specific compounds were identified 3-methyl-1-butanol and eugenol. The former resulted from reduction of 3-methylbutanal which can be synthetized from leucine via Ehrlich pathway or Strecker degradation. The presence of alcohol indicates strong activity of alcohol-dehydrogenase [5]. The aldehyde and its corresponding alcohol were already identified in sourdough or bread and alcohol formation is considered as the result of yeast action. y Eugenol is a simple phenolic compound, formed via the general phenylpropanoid pathway, including the conversion of coniferyl alcohol and the action of specific enzymes [6]. The richness in lignin of bran suggested a possible deviation of the general way toward eugenol. It is possible that its formation is a defence mechanism due to pathogens attack. Other compounds like acetophenone and derived components have been found in brans (data not shown) with feruloyl-coA as a precursor and their presence was also described in a context of plant defence. The production of eugenol is positive because it shows antimicrobial activity against fungi (Fusarium, Penicillium or Aspergillus sp) contaminating wheat. Moreover, as this compound, is found only in bran, it can play the role of a marker of bran addition in flour. Concerning the odour quality of compounds present in all fractions, the majority was characterized by green, vegetal aroma and are rather pleasant with the exception of acetic acid, 1-pentanol and 1-hexanol that are perceived as pungent. The presence of limonene can improve the sensorial attribute with citrus odour. Aromatic profile of wheat flour and bran fractions Aromatic profile of wheat flour and bran fractions Aromatic profile of wheat flour and bran fractions In the flour, the more represented compounds were limonene, 1-hexanol and nonanal followed by 2-heptanone. In the brans, limonene is clearly the major compound, but 1-hexanol and 2-heptanone were also identified. In general, the main contributors to the wheat flour aroma are the compounds derived from lipid peroxidation [3] and the amount of these compounds changed with the progress of reaction: the longer the time before analysis, the greater the risk of oxidation causing volatile profile modification. In their study Xu et al. [3] found that 1-hexanol was the major compound of the flour produced from common wheat grains followed by hexanal, and nonanal. Hexanol is produced by the action of alcohol dehydrogenase from hexanal. This latter is the main volatile product from the lipoxygenase activity and autoxidation degradation of linoleic acid. Nonanal is produced mostly from oleic acid and was strongly represented in flour. A high quantity of these compounds (aldehydes and alcohols) is an indication of major extend of lipid oxidation. In our fractions, their amounts remain relatively low suggesting a good preservation of the fractions. g p 2-Heptanone is formed by β-oxidation from octanoic acid in mitochondria of entire cell plants or by the action of fungi as Penicillium sp. Its higher presence in coarse brans could be due to the less intensive cell degradation. This hypothesis agrees with the presence of hexanoic acid, another product of β-oxidation of octanoic acid mainly in brans fractions. 2-Pentylfuran was only detected in brans but was not specific of these fractions because it has been already found in flour [3]; this component is formed via auto-oxidation of linoleic acid. The strong amount of (R)-limonene specifically in the brans (50% of volatile compounds) was unexpected. However, limonene and other terpenes were evidenced in flour [3] and in derived product as bread. Their presence could be explained by the contamination by other plants which could be cropped with wheat grains. Limonene is also the major component of insecticide, fungicide and acaricide used in organic farming for vegetables. Then, a cross over contamination could be also evocated to explain the high amount of this compound in the flour and particularly in the bran fractions which are constituted to the more external layer of grains. Results and discussion First, it is important to highlight that the HS-SPME method allowed to well extract the most volatile compounds compared to liquid-liquid extraction (data not shown). Numerous compounds due to lipid oxidation such as 1-pentanol, hexanol, 1-octen-3-ol, 2-heptanone, hexanal First, it is important to highlight that the HS-SPME method allowed to well extract the most volatile compounds compared to liquid-liquid extraction (data not shown). First, it is important to highlight that the HS-SPME method allowed to well extract the most volatile compounds compared to liquid-liquid extraction (data not shown). Numerous compounds due to lipid oxidation such as 1-pentanol, hexanol, 1-octen-3-ol, 2-heptanone, hexanal and nonanal were present in the 3 fractions (Table 1) but in different rather high amounts. p q q ( ) Numerous compounds due to lipid oxidation such as 1-pentanol, hexanol, 1-octen-3-ol, 2-heptanone, hexanal and nonanal were present in the 3 fractions (Table 1) but in different rather high amounts. Table 1: Aroma compounds of wheat grains milling fractions and their semi-quantification estimated using 2-heptanol as internal standard. Aroma Compound (mg/kg dry basis) LRI Flour Fine bran Coarse bran Odour qualitya Hexanal 1106 0.094± 0.003 0.057±0.009 0.093±0.009 Fresh green 2-Heptanone 1220 0.110±0.075 0.64±0.09 1.37±0.20 Cheese,fruity, (R)-Limonene 1227 0.504±0.135 4.93±0.55 4.64±0.44 Citrus, peely 3-Methyl-1-Butanol 1254 ND ND 0.127±0.004 Pleasant, fruity, brandy 2-Pentyl-furan 1263 ND 0.679±0.027 0.427±0.015 Beany, nut 1-Pentanol 1288 0.086±0.028 0.057±0.020 ND Pungent, bready 1-Hexanol 1388 0.441±0.063 0.905±0.039 0.936±0.033 Pungent, fruity Nonanal 1417 0.262±0.079 0.035±0.006 0.017±0.003 Aldehydic, citrus Acetic Acid 1481 0.049±0.005 0.090±0.004 0.066±0.003 Sour, acetic 1-Octen-3-ol 1487 0.051±0.006 0.104±0.007 0.059±0.004 Earthy, fungal 2-Ethylhexanol 1525 0.089±0.012 ND ND Citrus fresh floral Hexanoic Acid 1889 tr 0.156±0.009 0.087±0.005 Sour, fatty, cheese Benzyl Alcohol 1919 0.052±0.009 0.051±0.007 ND Sweet, floral, fruity Eugenol 2229 ND 0.134±0.008 0.120±0.005 Spicy, clove like, wood Total 1.799±0.521 7.843±0.845 7.944±0.738 aAll odour qualities collected at: http://www.thegoodscentscompany.com/. ND non detected e 1: Aroma compounds of wheat grains milling fractions and their semi-quantification estimated heptanol as internal standard. Table 1: Aroma compounds of wheat grains milling fractions and their semi-quantification estimated using 2-heptanol as internal standard. Quantitatively, the flour was less rich in volatile compounds (around to 2mg/kg dry basis) than fine and coarse brans (around 8mg/kg dry basis). As derived product of lipid represented the major part of volatiles components, it can be related to the lowest lipid content of flour compared to fine and coarse brans, 1.6, 5.3 and 4.7 %, respectively. 2 Aromatic profile of wheat flour and bran fractions The addition of bran in flour should not have a detrimental influence because numerous compounds are common to the flour and the two specific compounds have a pleasant odour. However, the oxidation reaction could be more important during storage in relation to the higher lipid content of brans and shelf life of product revaluated in functions of bran addition. 3 P. Chalier et al. P. Chalier et al. Conclusion Numerous volatile compounds coming from lipid oxidation are present in all milling fractions. The amount of volatile components is higher in brans than in flour in relation to the strongest lipids content of bran. Limonene is found in great quantity, surpassing all the other components, in particular in brans. Its origin should be investigated. The specific bran compounds are pleasant and should positively impact flour sensory properties. Moreover, eugenol may provide a specific marker of bran addition into flour. Its presence or that of other phenylpropanoids need to be checked on a large number of samples. p p ; 5. Smit B. A., Engel W.J.M., Smit G. 2009, Branched chain aldehydes: production and breakdown pathways and relevance for flavor in foods, Applied Microbiol. Biotechnol. 2009;81:987-999. 1. Hemery Y., Rouau, X., Lullien-Pellerin V., Barron, C., Abecassis, J. Dry processes to develop wheat fractions and products with enhanced nutritional quality. J. Cereal Science. 2007;46(3):327–347. y Wakayama T., Ito Y., Sakai K., Miyake M., Shibata E., Ohno H. et al Comprehensive review of 2ethyl-hexanol as r pollutant. J. Occup Health. 2019;61:19–35. , pp ; 6. Vogt T. Phenylpropanoid Biosynthesis. Molecular Plant, 2010;3(1):2-20. 2. Apprich, S., Ö. Tirpanalan, J. Hell, M. Reisinger, S. Böhmdorfer, S. Siebenhandl-Ehn, S. Novalin and W. Kneifel. Wheat bran-based biorefinery 2: Valorization of products. LWT - Food Science and Technology.2014;56(2): 222-231. 3. Xu, J., Zhang, W., Adhikari, K., & Shi, Y. C. Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats J Cereal Science 2017;75: 77–83 1. Hemery Y., Rouau, X., Lullien-Pellerin V., Barron, C., Abecassis, J. Dry processes to develop wheat fractions and products with enhanced nutritional quality. J. Cereal Science. 2007;46(3):327–347. 2. Apprich, S., Ö. Tirpanalan, J. Hell, M. Reisinger, S. Böhmdorfer, S. Siebenhandl-Ehn, S. Novalin and W. Kneifel. Wheat bran-based biorefinery 2: Valorization of products. LWT - Food Science and Technology.2014;56(2): 222-231. 3. Xu, J., Zhang, W., Adhikari, K., & Shi, Y. C. Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats. J. Cereal Science. 2017;75: 77–83. 4. Wakayama T., Ito Y., Sakai K., Miyake M., Shibata E., Ohno H. et al Comprehensive review of 2ethyl-hexanol as an indoor pollutant. J. Occup Health. 2019;61:19–35. 5. Smit B. A., Engel W.J.M., Smit G. 2009, Branched chain aldehydes: production and breakdown pathways and relevance for flavor in foods, Applied Microbiol. Biotechnol. 2009;81:987-999. 6 Vogt T Phenylpropanoid Biosynthesis Molecular Plant 2010;3(1):2-20 products with enhanced nutritional quality. J. Cereal Science. 2007;46(3):327–347. 2. Apprich, S., Ö. Tirpanalan, J. Hell, M. Reisinger, S. Böhmdorfer, S. Siebenhandl-Ehn, S. Novalin and W. Kneifel. Wheat bran-based biorefinery 2: Valorization of products. LWT - Food Science and Technology.2014;56(2): 222-231. 3. Xu, J., Zhang, W., Adhikari, K., & Shi, Y. C. Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats. J. Cereal Science. 2017;75: 77–83. 4 Wakayama T Ito Y Sakai K Miyake M Shibata E Ohno H et al Comprehensive review of 2ethyl hexanol as an References 1. Hemery Y., Rouau, X., Lullien-Pellerin V., Barron, C., Abecassis, J. Dry processes to develop wheat fractions and products with enhanced nutritional quality. J. Cereal Science. 2007;46(3):327–347. 2. Apprich, S., Ö. Tirpanalan, J. Hell, M. Reisinger, S. Böhmdorfer, S. Siebenhandl-Ehn, S. Novalin and W. Kneifel. Wheat bran-based biorefinery 2: Valorization of products. LWT - Food Science and Technology.2014;56(2): 222-231. 3. Xu, J., Zhang, W., Adhikari, K., & Shi, Y. C. Determination of volatile compounds in heat-treated straight-grade flours f l d h t J C l S i 2017 75 77 83 4
https://openalex.org/W4385995433	https://zenodo.org/records/8265939/files/45308.pdf	English	null	Poisoning with methanol and other non-beverage alcohols - an analysis of the issue based on hospitalizations in department of toxicology	Journal of Education, Health and Sport	2,023	cc-by	3,909	SOSNOWSKA, Weronika, BRZOZOWSKA, Aleksandra, TCHÓRZ, Michał, RADONIEWICZ–TCHÓRZ, Anna, TKACZYK, Rafał, TOMCZYK, Jakub, ŚWIĄTEK, Gabriela, TRUSZ, Kornelia and WANAT, Iwona. Poisoning with methanol and other non- beverage alcohols - an analysis of the issue based on hospitalizations in department of toxicology. Journal of Education, Health and Sport. 2023;45(1):11-21. eISSN 2391-8306. http://dx.doi.org/10.12775/JEHS.2023.45.01.001 https://apcz.umk.pl/JEHS/article/view/45308 https://zenodo.org/record/8265939 SOSNOWSKA, Weronika, BRZOZOWSKA, Aleksandra, TCHÓRZ, Michał, RADONIEWICZ–TCHÓRZ, Anna, TKACZYK, Rafał, TOMCZYK, Jakub, ŚWIĄTEK, Gabriela, TRUSZ, Kornelia and WANAT, Iwona. Poisoning with methanol and other non- beverage alcohols - an analysis of the issue based on hospitalizations in department of toxicology. Journal of Education, Health and Sport. 2023;45(1):11-21. eISSN 2391-8306. http://dx.doi.org/10.12775/JEHS.2023.45.01.001 https://apcz.umk.pl/JEHS/article/view/45308 https://zenodo.org/record/8265939 The journal has had 40 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of 17.07.2023 No. 32318. Has a Journal's Unique Identifier: 201159. Scientific disciplines assigned: Physical Culture Sciences (Field of Medical sciences and health sciences); Health Sciences (Field of Medical Sciences and Health Sciences). Punkty Ministerialne z 2019 - aktualny rok 40 punktów. Załącznik do komunikatu Ministra Edukacji i Nauki z dnia 17.07.2023 Lp. 32318. Posiada Unikatowy Identyfikator Czasopisma: 201159. Przypisane dyscypliny naukowe: Nauki o kulturze fizycznej (Dziedzina nauk medycznych i nauk o zdrowiu); Nauki o zdrowiu (Dziedzina nauk medycznych i nauk o zdrowiu). © The Authors 2023; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 21.07.2023. Revised:10.08.2023. Accepted: 18.08.2023. Published: 21.08.2023. Jakub Tomczyk Clinical Provincial Hospital No. 2 St. Queen Jadwiga in Rzeszów Poisoning with methanol and other non-beverage alcohols - an analysis of the issue based on hospitalizations in department of toxicology Weronika Sosnowska https://orcid.org/0009-0003-4608-5521 weronika.sosnowska07@gmail.com Stefan Wyszyński Regional Specialist Hospital, Lublin, Poland. Aleksandra Brzozowska https://orcid.org/0009-0008-7687-1140 brzozola@onet.eu Stefan Wyszyński Regional Specialist Hospital, Lublin, Poland. Michał Tchórz https://orcid.org/0000-0002-1308-099X tchorz.michal@gmail.com Toxicology Clinic, Medical University, Lublin, Poland. Anna Radoniewicz–Tchórz https://orcid.org/0009-0009-9055-8895 anna.radoniewicz@gmail.com Stefan Wyszyński Regional Specialist Hospital, Lublin, Poland. Rafał Tkaczyk https://orcid.org/0009-0001-0542-3374 rafaltkaczyk@wp.pl District Specialist Hospital in Stalowa Wola, Poland. 11 11 Jakub Tomczyk https://orcid.org/0009-0008-0586-5798 jjtomczyk15@gmail.com Stefan Wyszyński Regional Specialist Hospital, Lublin, Poland. Gabriela Świątek https://orcid.org/0009-0004-8537-6249 gabriela.swiatek21@gmail.com District Specialist Hospital in Stalowa Wola, Poland. Kornelia Trusz https://orcid.org/0009-0002-6291-6447 kornelia.trusz@gmail.com University Clinical Hospital named after Fryderyk Chopin in Rzeszów Iwona Wanat https://orcid.org/0009-0003-7125-3611 iw.wanat@gmail.com Clinical Provincial Hospital No. 2 St. Queen Jadwiga in Rzeszów ABSTRACT Introduction: Poisonings with non-beverage alcohols remain a significant problem in contemporary toxicology. Due to the large quantity and severe course of intoxications with methanol, the authors of this study focused primarily on poisonings with this substance. Methanol is widely used in industry. Its toxicity is attributed to its metabolites - formaldehyde and formic acid. Poisonings with methanol often lead to the patient's death due to a wide range of complications associated with progressive metabolic acidosis. Material and Methods: The study is of a retrospective nature. It involved the analysis of medical records of patients admitted to the department of toxicology in 2022 after the consumption of non-beverage alcohols, with a particular focus on methanol. Data on the number of individuals admitted due to non-beverage alcohol intoxication between 2018 and 2022 was also analyzed. Results: Over the last 5 years (2018-2022), 36 individuals were hospitalized in the Clinical Toxicological-Cardiological Department due to non-beverage alcohol poisoning, with 16 (44.4%) cases involving methanol. In the year 2022, the department admitted 10 individuals 12 aged 18 to 68, and 5 (50%) of them consumed methanol. Among the hospitalized, 90% were male, and 60% came from rural areas. Deliberate intoxications accounted for 70% of the cases. Four hospitalizations (40%) resulted in patient death. All deaths were associated with methanol intoxication. Conclusions: Non-beverage alcohol is often used by people with alcohol dependence syndrome. Among non-beverage alcohols, patients most often reached for methanol. Its consumption is associated with a high mortality rate and should be taken very seriously and treated immediately. Keywords: methanol; poisoning; intoxication Introduction Among non-beverage alcohols, the following should be mentioned: isopropyl alcohol, methanol (methyl alcohol), ethylene glycol, diethylene glycol, and propylene glycol [1]. Due to the exceptionally severe nature of methanol poisonings, the authors primarily focus on the issue of intoxication with this non-beverage alcohol. Methanol has a wide range of industrial applications, making it relatively accessible for intentional poisonings. It is used in the production of antifreeze, cleaning fluids, dyes, aviation fuels, and photocopier fluids [2]. In some cases, especially in developing countries, food-grade alcohol (ethanol) can be contaminated with methanol [3]. The toxicity of methanol is attributed to the formation of formic acid as a result of metabolic transformations [4]. Formic acid causes metabolic acidosis and, by inhibiting the cytochrome oxidase complex, also leads to lactic acidosis [5]. The individual course of methanol intoxication varies and depends, among other factors, on the pool of folates, which play a role in the elimination of formic acid from the body [6]. Methanol itself induces central nervous system depression, leading to decreased cardiac output, stroke volume, and blood pressure reduction [7]. A characteristic symptom of methanol poisoning is visual impairment [8]. It is estimated that visual loss can occur with as little as 4 to 10 ml of a 40% solution of methanol [9]. It has been demonstrated that the severity of ocular changes and overall toxicity of methanol correlate with the degree of 13 metabolic acidosis [10]. The wide spectrum of accompanying symptoms in methanol poisoning results from the increasing metabolic acidosis caused by lactate accumulation [5]. Particularly unfavorable prognostic factors include decreased pH (especially <7.0), coma, and seizures [11]. The lethal dose of methanol is 15-30 ml of a 40% solution of methanol. Material and Methods The conducted study has a retrospective nature. The subjects of the study were patients hospitalized in 2022 at the Clinical Department of Toxicology and Cardiology of the Provincial Specialist Hospital named after Stefan Cardinal Wyszyński in Lublin, due to the consumption of non-beverage alcohols. The data was collected based on the analysis of medical records of the patients. During the analysis of medical records, various information such as gender, age, place of residence, the nature of intoxication, length of patients' stay in the department, and the quarter of the year in which the patient was admitted to the hospital were taken into consideration. Additionally, a specific group of patients intoxicated with methanol was isolated, and the laboratory parameters measured at admission, such as blood pH and methanol concentration in the blood, were analyzed. Moreover, the number of intoxications with non-beverage alcohols, with a particular focus on methanol, was compared over the last 5 years (2018-2022). Results Among the participants, 4 (40%) were from urban areas, while 6 (60%) were from rural areas. In 7 (70%) cases, the intake of the toxic substance was incidental. Unintentional poisonings were caused by alcohol craving and mood-enhancing attempt among people with alcohol dependence syndrome or mistakenly consuming toxic substances poured into containers previously used for beverage packaging, such as mineral water or fruit juice. A diagnosis of alcohol addiction syndrome was made for 6 (60%) individuals. Throughout the year, 3 patients (30%) were admitted to the department in each of the second, third, and fourth quarters, while the lowest number of admissions occurred in the first quarter (10%). The average length of hospital stay was approximately 6 days, with the shortest stay being for patients discharged on the day of admission and the longest stay lasting 32 days. 4 (40%) hospitalizations resulted in patient deaths. All fatalities were associated with methanol intoxication. Out of the 10 cases, 5 (50%) individuals consumed methanol. All of In the year 2022, a total of 10 individuals were hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to consumption of non-beverage alcohols. The age of the patients included in the study ranged from 18 to 68 years. Male intoxications accounted for a significant majority (90%). Among the participants, 4 (40%) were from urban areas, while 6 (60%) were from rural areas. In 7 (70%) cases, the intake of the toxic substance was incidental. Unintentional poisonings were caused by alcohol craving and mood-enhancing attempt among people with alcohol dependence syndrome or mistakenly consuming toxic substances poured into containers previously used for beverage packaging, such as mineral water or fruit juice. A diagnosis of alcohol addiction syndrome was made for 6 (60%) individuals. Throughout the year, 3 patients (30%) were admitted to the department in each of the second, third, and fourth quarters, while the lowest number of admissions occurred in the first quarter (10%). The average length of hospital stay was approximately 6 days, with the shortest stay being for patients discharged on the day of admission and the longest stay lasting 32 days. 4 (40%) hospitalizations resulted in patient deaths. All fatalities were associated with methanol intoxication. Out of the 10 cases, 5 (50%) individuals consumed methanol. Results In the years 2018-2022, a total of 36 individuals were admitted to the department after consuming non-beverage alcohols, of which 16 (44.4%) consumed methanol. The numerical distribution of hospitalizations in each year is presented in Chart 1. 14 Chart 1. The number of people hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to non-beverage alcohol poisoning with a breakdown of methanol cases in the years 2018-2022. Chart 1. The number of people hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to non-beverage alcohol poisoning with a breakdown of methanol cases in the years 2018-2022. In the year 2022, a total of 10 individuals were hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to consumption of non-beverage alcohols. The age of the patients included in the study ranged from 18 to 68 years. Male intoxications accounted for a significant majority (90%). Among the participants, 4 (40%) were from urban areas, while 6 (60%) were from rural areas. In 7 (70%) cases, the intake of the toxic substance was incidental. Unintentional poisonings were caused by alcohol craving and mood-enhancing attempt among people with alcohol dependence syndrome or mistakenly consuming toxic substances poured into containers previously used for beverage packaging, such as mineral water or fruit juice. A diagnosis of alcohol addiction syndrome was made for 6 (60%) individuals. Throughout the year, 3 patients (30%) were admitted to the department in each of the second, third, and fourth quarters, while the lowest number of admissions occurred in the first quarter (10%). The average length of hospital stay was approximately 6 days, with the shortest stay being for patients discharged on the day of admission and the longest stay lasting 32 days. 4 (40%) hospitalizations resulted in patient deaths. All fatalities were associated with methanol intoxication. Out of the 10 cases, 5 (50%) individuals consumed methanol. All of Chart 1. The number of people hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to non-beverage alcohol poisoning with a breakdown of methanol cases in the years 2018-2022. In the year 2022, a total of 10 individuals were hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to consumption of non-beverage alcohols. The age of the patients included in the study ranged from 18 to 68 years. Male intoxications accounted for a significant majority (90%). Results All of In the year 2022, a total of 10 individuals were hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to consumption of non-beverage alcohols. The age of the patients included in the study ranged from 18 to 68 years. Male intoxications accounted for a significant majority (90%). Among the participants, 4 (40%) were from urban areas, while 6 (60%) were from rural areas. In 7 (70%) cases, the intake of the toxic substance was incidental. Unintentional poisonings were caused by alcohol craving and mood-enhancing attempt among people with alcohol dependence syndrome or mistakenly consuming toxic substances poured into containers previously used for beverage packaging, such as mineral water or fruit juice. A diagnosis of alcohol addiction syndrome was made for 6 (60%) individuals. Throughout the year, 3 patients (30%) were admitted to the department in each of the second, third, and fourth quarters, while the lowest number of admissions occurred in the first quarter (10%). The average length of hospital stay was approximately 6 days, with the shortest stay being for patients discharged on the day of admission and the longest stay lasting 32 days. 4 (40%) hospitalizations resulted in patient deaths. All fatalities were associated with methanol intoxication. Out of the 10 cases, 5 (50%) individuals consumed methanol. All of In the year 2022, a total of 10 individuals were hospitalized at the Clinical Department of Toxicology and Cardiology in Lublin due to consumption of non-beverage alcohols. The age of the patients included in the study ranged from 18 to 68 years. Male intoxications accounted for a significant majority (90%). Among the participants, 4 (40%) were from urban areas, hil 6 (60%) f l I 7 (70%) th i t k f th t i b t 15 these cases involved male patients. 80% of them were residing in rural areas. In 3 cases (60%), the methanol consumption was intentional, with one of them being a suicide attempt. Table 1 presents the laboratory parameters measured upon admission, including blood pH and methanol concentration. As a result of methanol consumption, 4 individuals (80%) died. Table 1. Information regarding measured methanol concentrations in the blood and blood pH values in patients intoxicated with methanol. PARAMETER AVERAGE RANGE OF VALUES (MIN-MAX) STANDARD RANGE Methanol concentration (mg/dl) 227,63 60-473.52 - Blood pH 7,12 6,94-7,29 7,35–7,45 Discussion Among non-beverage alcohol poisonings, methanol poisoning is the most frequently observed and the most serious. Therefore, this study primarily focuses on methanol intoxication. The examined group of individuals reflects the issue of non-beverage alcohol poisonings only among those admitted to the Clinical Toxicology and Cardiology Department at WSS Hospital in Lublin. However, the results of this study allow for the observation of certain patterns regarding the type of substance consumed and the individuals affected by poisoning. In recent years, there has been an upward trend in non-beverage alcohol poisonings, particularly those involving methanol. The age structure of patients admitted to the department varies significantly, ranging from 18 to 68 years old. It is difficult to identify a specific age group that is more susceptible to methanol exposure. However, it can be noticed that poisonings occur more frequently in rural areas. Out of 36 people admitted to the Clinical Toxicology and Cardiology Department at WSS Hospital in Lublin between 2018 and 2022, 16 individuals (44.4%) were there due to methanol poisoning. Significantly larger proportion of poisonings, about 90%, were among males (similar results were obtained by Ahmed F et al. [12]), with 60% of them being diagnosed with alcohol dependence syndrome. Alcohol dependence syndrome is more common in males [13] and is also more likely to lead to death and have a higher disability- 16 adjusted life years (DALYs) rate, which represents the number of years lost due to disability or premature death in the male population [13]. Given the increasing number of non-beverage poisonings, including methanol, one might ask why do poisonings occur? Methanol is cheaper and more readily available than ethanol, and some people use it in the production of homemade alcohol, which they then sell at a favorable price to individuals addicted to alcohol as an alcohol substitute [14]. This homemade alcohol may contain pure methanol, methanol diluted with water, or methanol with a mixture of ethanol [15]. At the beginning of the COVID-19 pandemic, there was a misconception among people that methanol had antiviral properties. In Iran, more than 1000 people consumed methanol to fight the virus, and over 300 people died from poisoning [16]. It is also common for methanol to be stored in non-original containers, such as being poured into bottles from water, beverages, or ethanol. This can lead to accidental consumption and poisoning. Conclusions 1. Non-beverage alcohol is often used by people with alcohol dependence syndrome. 2. Methanol poisoning occurs most frequently. 3. The reasons for methanol consumption vary, and it is essential to quickly identify the poisoning and implement appropriate treatment due to the serious symptoms that can result in the patient's death. 4. The metabolites of methanol are toxic, not methanol itself, which is why inhibiting alcohol dehydrogenase is an effective treatment for poisoning. 5. The method of extracorporeal elimination of choice is the intermittent hemodialysis procedure, during which both the methanol itself and its metabolites are removed from the patient's vascular compartment. Discussion Methanol itself is not cytotoxic, but during its metabolism, formaldehyde and formic acid are produced, which can disrupt oxidative metabolism by inhibiting the cytochrome oxidase mechanism [16]. Symptoms of poisoning can include coma and respiratory arrest; hence, patients with severe poisoning require intubation and mechanical ventilation [17]. The most critical aspect of treatment for patients with suspected or confirmed methanol poisoning is the blockade of alcohol dehydrogenase (ADH), which prevents the formation of toxic metabolites [18] and intermittent hemodialysis used to eliminate both methanol and its metabolites, in addition, to compensate for blood gas and electrolyte abnormalities. The intermittent hemodialysis procedure is performed in patients with serum methanol levels above 50 mg/dl and/or with severe metabolic acidosis, deterioration of general condition despite adequate treatment, severe electrolyte disturbances refractory to conservative treatment, or acute renal failure. Substances that block ADH are: ethanol, fomepizole, abacavir, and H2 receptor blockers [16]. The most commonly used substances are ethanol and fomepizole [19]. Ethanol: 1 ml/kg of 96% alcohol diluted fivefold as the loading dose, 0.16 ml/kg/h as the maintenance dose orally or via NGT, or 10% ethanol: 10 ml/kg intravenously as the loading dose, then 1 ml/kg/h until achieving an ethanol serum level of 150 mg/dl. Patients receiving intravenous (IV) ethanol require admission to an intensive care unit (ICU) due to numerous 17 17 adverse effects caused by ethanol, such as hypotension, respiratory depression, erythema, hypoglycemia, pancreatitis, and gastritis [16]. Fomepizole: (4-methylpyrazole) at a dose of 15 mg/kg as the loading dose, 10 mg/kg as the maintenance dose every 12 hours for up to 4 doses, and then 15 mg/kg every 12 hours until the methanol concentration in the blood drops below 25 mg/dl. Fomepizole does not require monitoring as in the case of ethanol infusion since it does not cause intoxication and is associated with fewer side effects. However, it is an expensive drug [16]. Antidote administration is recommended as soon as possible, preferably during pre-hospital care at the scene of the incident, if possible. Potential misuse of oral ethanol is rare in cases of methanol poisoning, and the benefits of early antidote administration outweigh the potential risk of side effects [20]. Ethanol is used as the primary antidote in toxicology departments. Special clinical situations oblige the use of fomepizole [21]. Informed Consent Statement Not applicable. Author Contributions Conceptualization and methodology:W.S., A.B., A.R-T. and M.T.; formal analysis, data curation , writing: W.S., A.B., A.R-T., J.T.,I.W., K.T., G.Ś., R.T.; supervision M.T. All authors have read and agreed to the published version of the manuscript. 18 Conflicts of Interest The authors declare no conflict of interest. Institutional Review Board Statement Not applicable. Not applicable. Funding This research received no external funding. Institutional Review Board Statement Not applicable. 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https://openalex.org/W2978422643	https://www.mdpi.com/2072-6643/11/10/2371/pdf?version=1571069023	English	null	Dietary Intake of Green Nut Oil or DHA Ameliorates DHA Distribution in the Brain of a Mouse Model of Dementia Accompanied by Memory Recovery	Nutrients	2,019	cc-by	7,604	Keywords: DHA; green nut oil; Alzheimer’s disease; dementia; SAMP8 mouse; DESI-IMS; memory eﬃciency nutrients nutrients Dietary Intake of Green Nut Oil or DHA Ameliorates DHA Distribution in the Brain of a Mouse Model of Dementia Accompanied by Memory Recovery Emiko Takeyama 1,2,†, Ariful Islam 3,†, Nakamichi Watanabe 1,2, Hiroe Tsubaki 4, Masako Fukushima 2, Md. Al Mamun 3, Shumpei Sato 3,5, Tomohito Sato 3,5, Fumihiro Eto 3,6, Ikuko Yao 5,6, Takashi K. Ito 3,5, Makoto Horikawa 3,5 and Mitsutoshi Setou 3,5,7,* 1 Department of Food Science and Nutrition, Graduate School of Human Life Sciences, Showa Women’s University, 1-7-57 Taishido, Setagaya-ku, Tokyo 154-8533, Japan; takeyama@swu.ac.jp (E.T.); nakamich@swu.ac.jp (N.W.) 1 Department of Food Science and Nutrition, Graduate School of Human Life Sciences, Showa Women’s University, 1-7-57 Taishido, Setagaya-ku, Tokyo 154-8533, Japan; 2 Institute of Women’s Health Sciences, Showa Women’s University, 1-7-57 Taishido, Setagaya-ku, Tokyo 154-8533, Japan; m2-fukushima@outlook.jp 3 Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan; ariful222222@gmail.com (A.I.); amamun5245@gmail.com (M.A.M.); shumpe.sato@gmail.com (S.S.); titon0620@gmail.com (T.S.); etofmhr@gmail.com (F.E.); itotakakun77777@gmail.com (T.K.I.); mh.alkerus@gmail.com (M.H.) 4 The Institute of Statistical Mathematics, 10-3 Midori-cho, Tachikawa-si, Tokyo 190-8562, Japan; tsubaki@ism.ac.jp 3 Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan; ariful222222@gmail.com (A.I.); amamun5245@gmail.com (M.A.M.); shumpe.sato@gmail.com (S.S.); titon0620@gmail.com (T.S.); etofmhr@gmail.com (F.E.); itotakakun77777@gmail.com (T.K.I.); mh.alkerus@gmail.com (M.H.) g g g 4 The Institute of Statistical Mathematics, 10-3 Midori-cho, Tachikawa-si, Tokyo 190-8562, Japan; tsubaki@ism.ac.jp jp 5 International Mass Imaging Center, Hamamatsu University School of Medicine, 1-20-1 Handayama Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan; yaoik5@gmail.com ernational Mass Imaging Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, 5 International Mass Imaging Center, Hamamatsu University School of Medicin Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan; yaoik5@gmail.com Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan; yaoik5@gmail.com 6 Department of Optical Imaging, Institute for Medical Photonics Research, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku Hamamatsu, Shizuoka 431-3192, Japan p 7 Department of Systems Molecular Anatomy, Institute for Medical Photonics Research, Preeminent Medical Photonics Education & Research Center, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan 7 Department of Systems Molecular Anatomy, Institute for Medical Photonics Research, Preeminent Medical Photonics Education & Research Center, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan * Correspondence: setou@hama med ac jp; Tel : 053 435 2086; Fax: 053 435 2468 y g J p Correspondence: setou@hama-med.ac.jp; Tel.: 053-435-2086; Fax: 053-435-2468 y g * Correspondence: setou@hama-med.ac.jp; Tel.: 053-435-2086; Fax: 053-435-2468 p jp These authors contributed equally to this work. † These authors contributed equally to this work. nutrients nutrients 1. Introduction Docosahexaenoic acid (DHA), one of the most important omega-3 (ω -3) polyunsaturated fatty acid (PUFA) found in the brain, is essential for the development and function of the brain [1,2]. Dietary supplementation of DHA has attracted signiﬁcant interest in the last few decades for its several health beneﬁts [3]. DHA has been linked to a lower risk of several prevalent diseases, including cancer, cardiovascular disorders, and Alzheimer’s disease (AD) [4–6]—one of the most common types of dementia and the most common neurodegenerative disorder in elderly people. In 2018, the number of patients aﬀected by AD in the U.S. alone was estimated to be 5.7 million [7]. The major pathological hallmarks of AD are the deposition of amyloid β (Aβ) plaques and hyperphosphorylated tau in the brain [8]. In addition, Aβ is the main determinant of neuronal loss and synaptic disruption in the development of dementia [9]. p Previous studies have reported decreased levels of linoleic acid (LA; C18:2), oleic acid (OA; C18:1), arachidonic acid (AA; C20:4), eicosapentaenoic acid (EPA; C20:5), docosapentaenoic acid (DPA; C22:5), DHA (C22:6), and DHA-containing phosphatidylcholines in the brain of patients with dementia [2,10,11]. DHA supplementation increases levels of DHA in the brain and alleviates the Aβ load, tau phosphorylation, neuroinﬂammation, and neuronal apoptosis [12–15]. There is, however, a paucity of data on changes in the spatial distribution of free fatty acids (FFAs) in the brain after dietary intake of DHA. Thus, the present study was conducted in order to evaluate changes in the spatial distribution of DHA and other FFAs associated with dementia in the brain of senescence-accelerated mouse-prone 8 (SAMP8) mice—a mouse model of dementia, after dietary supplementation with DHA [16,17]. SAMP8 mice display many features of dementia, such as oxidative damage, degeneration of the brain stem, neuronal cell death, Aβ alternation, tau hyperphosphorylation, blood-brain barrier dysfunction, progressive learning, and memory deﬁcits [16,17]. Additionally, the eﬀects of green nut oil (GNO), also known as sacha inchi oil—oil extracted from the seeds of the evergreen climbing plant Plukenetia volubilis, were investigated in SAMP8 mice. GNO contains approximately 50% α-linolenic acid [18,19]. Human and other mammals can synthesize DHA from α-linolenic acid through a progressive series of enzymatic elongation and desaturation reactions [2,3]. Received: 19 July 2019; Accepted: 2 October 2019; Published: 4 October 2019 Abstract: Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has signiﬁcant health beneﬁts. Previous studies reported decreased levels of DHA and DHA-containing phosphatidylcholines in the brain of animals suﬀering from Alzheimer’s disease, the most common type of dementia; furthermore, DHA supplementation has been found to improve brain DHA levels and memory eﬃciency in dementia. Oil extracted from the seeds of Plukenetia volubilis (green nut oil; GNO) is also expected to have DHA like eﬀects as it contains approximately 50% α-linolenic acid, a precursor of DHA. Despite this, changes in the spatial distribution of DHA in the brain of animals with dementia following GNO or DHA supplementation remain unexplored. In this study, desorption electrospray ionization imaging mass spectrometry (DESI-IMS) was applied to observe the eﬀects of GNO or DHA supplementation upon the distribution of DHA in the brain of male senescence-accelerated mouse-prone 8 (SAMP8) mice, a mouse model of dementia. DESI-IMS revealed that brain DHA distribution increased 1.85-fold and 3.67-fold in GNO-fed and DHA-fed SAMP8 mice, respectively, compared to corn oil-fed SAMP8 mice. Memory eﬃciency in SAMP8 mice was also improved by GNO or DHA supplementation. In summary, this study suggests the possibility of GNO or DHA supplementation for the prevention of dementia. Keywords: DHA; green nut oil; Alzheimer’s disease; dementia; SAMP8 mouse; DESI-IMS; memory eﬃciency Nutrients 2019, 11, 2371; doi:10.3390/nu11102371 www.mdpi.com/journal/nutrients Nutrients 2019, 11, 2371 2 of 14 1. Introduction As a result of the well-known health beneﬁts of DHA, the continuing demand for DHA containing ﬁsh oil has exerted a substantial impact upon marine populations; thus, it has been suggested that mass scale ﬁshing is no longer sustainable [20]. GNO is expected to be a sustainable alternative source of DHA instead of ﬁsh oil DHA. Moreover, GNO is less expensive compared to DHA and does not require special instruments for extraction and puriﬁcation. Over the last few decades, imaging mass spectrometry (IMS) has become a powerful tool for the direct visualization, identiﬁcation, and analysis of biomolecules in proteomics and metabolomics [21,22]. By using IMS, several FFAs, phospholipids, peptides, and other metabolites have been identiﬁed in a number of tissues [23–26]. A recently developed IMS technique, known as desorption electrospray ionization imaging mass spectrometry (DESI-IMS), has emerged as a rapid, nondestructive, and powerful IMS technique for the detection of FFAs, phospholipids, proteins, drugs, metabolites, and other small molecules at µm scale resolution under ambient condition with the advantage of minimal sample preparation [27–29]. In this study, DESI-IMS was applied in order to analyze changes in the spatial distributions of FFAs, including DHA, in the brain of SAMP8 mice after supplementation with GNO or DHA. The Y-maze test was performed to investigate the eﬀects of GNO or DHA supplementation upon the behavioral phenotype of SAMP8 mice. 2.3. Group Size and Samples For behavioral analysis, group sizes were as follows: DHA, n = 10; GNO, n = 9; and CO, n = 9. For DESI-IMS analysis, group sizes were as follows: DHA, n = 3; GNO, n = 3; and CO, n = 3. For DESI-IMS analysis, three mice were selected from each group by stratiﬁed sampling. All nine mice taken for DESI-IMS analysis were divided into three experimental sets by blindly taking one mouse from each group for each experiment. 2.2. Chemicals and Reagents Fish oil DHA-46 and GNO were provided by Tama Biochemical Co., Ltd. (Tokyo, Japan) and NPO Arcoiris Naturaleza (Matsudo, Chiba, Japan), respectively. Corn oil was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). LC/MS grade methanol, 2-propanol, and ultrapure water were purchased from Wako Pure Chemical Industries (Osaka, Japan). OA, lysophosphatidylcholine (18:1), phosphatidylcholine (18:1/18:1), DHA, phosphatidylcholine (22:6/22:6), and sodium formate were purchased from Sigma-Aldrich (St. Louis, MO, USA). AA was purchased from Cayman Chemical (Ann Arbor, Michigan, MI, USA). 2.4. Tissue Preparation Following collection, the brains were rapidly frozen in powdered dry ice and stored at −80 ◦C. The tissues were mounted on a sample holder using optimal cutting temperature (OCT) compound (Sakura Finetek Japan, Tokyo, Japan) and sectioned at 10 µm thickness along the sagittal axis using a cryostat system (CM1950; Leica Biosystems, Wetzlar, Germany) at −20 ◦C on un-coated glass slides (Matsunami, Osaka, Japan, size: 76/26 mm; thickness: 0.8 to 1 mm) for DESI-IMS analysis. Hematoxylin and eosin (H&E) staining was used in order to facilitate histopathological examination. 2.1. Animal 2.1. Animal Thirteen weeks old male SAMP8 mice were purchased from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan) and reared in individual cages at 22 ± 2 ◦C with a light/dark cycle of 12 h (light 3 of 14 Nutrients 2019, 11, 2371 period: 7 am to 7 pm). Food and tap water were provided ad libitum. During the ﬁrst week of the preparatory rearing periods, the diet consisted of CRF-1 (Charles River Formula-1) pellets from Charles River International Laboratories, Inc. (Kanagawa, Japan). During the subsequent rearing period, the diet consisted primarily of AIN-93G (American Institute of Nutrition 93-G) from Oriental Yeast Co., Ltd., Tokyo, Japan. After the 1st week of preparatory rearing periods, the mice were divided into three groups. Group I (CO-fed) was comprised of SAMP8 mice that were fed a diet consisting of AIN-93G, in which soybean oil was replaced with 7% corn oil (CO). Group II (GNO-fed) was comprised of SAMP8 mice, fed on a diet consisting of AIN-93G, in which soybean oil was replaced with 7% GNO. Group III (DHA-fed) comprised of SAMP8 mice that were fed a diet consisting of AIN-93G, in which soybean oil was replaced with a mixture of 7% ﬁsh oil DHA-46 and CO (DHA-46:CO = 4:3). y p ( ) CO, GNO, and DHA-fed mice were reared for 14 weeks. After 14 h of fasting and 2 h of water deprivation on the last day of rearing, mice were euthanized by decapitation and dissected. Following removal, the brain samples were stored at −80 ◦C until further analysis. This study was approved by the Showa Women’s University Animal Research Committee and conducted according to the regulations of Showa Women’s University Animal Research Committee (ethical permission number: 17-04), and Animal Care and Use Committee of the Hamamatsu School of Medicine. 2.6. DESI-IMS Data Analysis MassLynx (Waters, Milford, MA, USA; version 4.1) software was used for data acquisition and processing, HDImaging (Waters, Milford, MA, USA; version 1.4) and IMAGE REVEAL (Shimadzu, Kyoto, Japan; version 1.0.1.8345) software was used for image analysis. IMAGE REVEAL was used particularly for the analysis of the intensity of each m/z at each pixel. 2.5. DESI-IMS Brain sections were kept at room temperature to dry before DESI-IMS acquisition. Mass spectra were acquired in negative ion modes. All experiments were performed using a DESI source attached to a quadrupole time-of-ﬂight (Q-TOF) mass spectrometer (Xevo G2-XS Q-TOF, Waters, Milford, MA, USA). The mass spectra were calibrated externally using sodium formate solution (500 µM) in a 90:10 ratio of 2-propanol: water (v/v), prior to measurement. The spray solvent (98:2; methanol: water, v/v) was delivered at a ﬂow rate of 2 µL/min using a solvent pump (ACQUITY UPLC Binary Solvent Manager, Waters, Milford, MA, USA). Ions from brain tissues were obtained in a mass range of m/z (mass-to-charge ratio) 100 to 1200 Da. All other parameters used in DESI-IMS are listed in Table 1. 4 of 14 Nutrients 2019, 11, 2371 Table 1. Parameters used for DESI-IMS analysis. DESI Ion Source Parameters Capillary Voltage −4 kV Source Temperature 100 ◦C Spray Impact angle 80◦ Solvent 98% Methanol (v/v) Solvent Flow Rate 2 µL/min Nebulizing N2 Gas Pressure 0.4 MPa DESI stage parameters Pixel size 100 µM Scanning speed (X axis) 200 µM/sec Data acquisition parameters Polarity −Ve m/z range 100 to 1200 Da Resolution 20000 Mass window 0.02 Da Acquisition rate 1 spectrum sec-1 Inlet voltage 4000 V With the exception of m/z 303.23 and 327.23, to assign all other candidate molecules corresponding to each targeted m/z, the Human Metabolome Database (http://www.hmdb.ca/spectra/ms/search) for mass accuracy and biological distribution was used alongside previous reports [30]. Tandem mass spectrometry (MS/MS) was performed in order to conﬁrm molecular ions against m/z 303.23 and 327.23 on the same instrument using collision energy 10 eV, source temperature 120 ◦C, capillary voltage 4 kV, and 98% methanol as solvent at a ﬂow rate 2 µL/min. To conﬁrm whether all detected molecular ions corresponded to fatty acids (FAs) were from FFAs or FA fragments from lipids, standard OA, OA containing lysophosphatidylcholine (LPC), OA containing phosphatidylcholine (PC), DHA, and DHA containing PC were analyzed using same instruments and parameters as mentioned in Table 1. 2.7. Mouse Behavior Behavioral tests were conducted using the Y-maze test, with slight modiﬁcations to the method reported by Yamada et al. [31]. Brieﬂy, mice were placed inside a Y-shaped device. Three arms (A, B, and C) contained objects of diﬀerent colors and shapes placed on the outer walls. Exploratory behavior of the mice was recorded. Y-maze trials were performed three times in total during the breeding period (at the age of 14 weeks, 22 weeks, and 27 weeks) for all mice in each group. Measurements were performed for 8 min. The arms that a mouse entered while freely exploring inside the maze were recorded in order as the "total arm entry number (spontaneous behavior amount)". The number of times a mouse selected diﬀerent arms three consecutive times was calculated as the "number of alternating behaviors". “Replacement behavior ratio" was determined as follows to assess spatial/short-term memory: ternating behavior rate (%) = number of alternating actions/(total arm entry number −2) × 100. Eﬃciency = log (alternating behavior rate/number of alternating actions). 5 of 14 Nutrients 2019, 11, 2371 2.8. Statistical Analysis All statistical analyses were performed using SPSS (version 16) and MS Excel (version 2019) software. All values are expressed as mean ± SEM (standard error of the mean). Diﬀerences were considered signiﬁcant with p values less than 0.05 (one-way ANOVA with Tukey′s multiple comparisons). IMAGE REVEAL (Shimadzu, Kyoto, Japan; version 1.0.1.8345) software was used to test the distribution pattern (Gaussian distribution or not) of DHA in the brain of SAMP8 mice. 3. Results 3.1. Detection of Target Peaks (m/z) from DESI-IMS Spectra DESI-IMS experiments in negative mode over 100–1200 m/z range were performed, and mass spectra from SAMP8 mouse brains were acquired in order to analyze changes in the lipoquality following GNO or DHA supplementation. Figure 1 indicates the representative mass spectra obtained from SAMP8 mouse brains, and Figure S1 depicts assigned peaks from DESI mass spectrum. Figure 1. Representative mass spectrum of DESI-IMS at negative ion mode over m/z range 100–1200 Da acquired from sagittal slices of senescence-accelerated mouse-prone 8 (SAMP8) mouse brains. CO-fed, GNO-fed, and DHA-fed indicate corn oil-fed SAMP8 mice, green nut oil-fed SAMP8 mice, and docosahexaenoic acid-fed SAMP8 mice, respectively. Figure 1. Representative mass spectrum of DESI-IMS at negative ion mode over m/z range 100–1200 Da acquired from sagittal slices of senescence-accelerated mouse-prone 8 (SAMP8) mouse brains. CO-fed, GNO-fed, and DHA-fed indicate corn oil-fed SAMP8 mice, green nut oil-fed SAMP8 mice, and docosahexaenoic acid-fed SAMP8 mice, respectively. In total, seven ions of interest were observed across the three groups with m/z 279.23, 281.25, 301.22, 303.23, 327.23, 329.25, and 331.27; these ions corresponded to LA (C18:2), OA (C18:1), EPA (C20:5), AA (C20:4), DHA (22:6), DPA (22:5), and adrenic acid (AdA; C22:4) respectively. With the exception of AdA, all other assigned FAs are associated with dementia. Corresponding molecular ions for m/z 303.23 and 327.23 were conﬁrmed by tandem mass spectrometry (Figure S2). All other candidates were assigned based on their m/z accuracy (Table 2) and previous reports [30]. 6 of 14 Nutrients 2019, 11, 2371 Table 2. m/z accuracy of targeted molecules. Table 2. m/z accuracy of targeted molecules. y g Free Fatty Acids Theoretical m/z Observed m/z Mass Accuracy (ppm) Linoleic acid (LA) 279.2330 279.2331 0.36 Oleic acid (OA) 281.2486 281.2487 0.36 Eicosapentaenoic acid (EPA) 301.2173 301.2170 1.00 Docosapentaenoic acid (DPA) 329.2486 329.2451 10.63 Adrenic acid (AdA) 331.2643 331.2639 1.21 Additionally, ﬁve lipid standards were analyzed using the same instruments and parameters which were used to acquire mass spectra from SAMP8 mouse brains. FFAs were detected eﬃciently by DESI-IMS, but no fragments for FAs from LPC (18:1), PC (18:1/18:1), and PC (22:6/22:6) were detected (Figure S3). 3.2. Eﬀects of Dietary Intake of GNO or DHA on Brain Distribution of FFAs 3.2. Eﬀects of Dietary Intake of GNO or DHA on Brain Distribution of FFAs After 14 weeks of GNO or DHA supplementation to SAMP8 mice, the distribution of LA, OA, EPA, AA, DHA, DPA, and AdA (Figure 2) was analyzed. After 14 weeks of GNO or DHA supplementation to SAMP8 mice, the distribution of LA, OA, EPA, AA, DHA, DPA, and AdA (Figure 2) was analyzed. Figure 2. Selected molecular ion (M-H)- images of free fatty acids in sagittal slices of SAMP8 mouse brains from DESI-IMS. LA: linoleic acid, OA: oleic acid, EPA: eicosapentaenoic acid, AA: arachidonic acid, DHA: docosahexaenoic acid, DPA: docosapentaenoic acid, AdA: adrenic acid. Figure 2. Selected molecular ion (M-H)- images of free fatty acids in sagittal slices of SAMP8 mouse brains from DESI-IMS. LA: linoleic acid, OA: oleic acid, EPA: eicosapentaenoic acid, AA: arachidonic acid, DHA: docosahexaenoic acid, DPA: docosapentaenoic acid, AdA: adrenic acid. Figure 2. Selected molecular ion (M-H)- images of free fatty acids in sagittal slices of SAMP8 mouse brains from DESI-IMS. LA: linoleic acid, OA: oleic acid, EPA: eicosapentaenoic acid, AA: arachidonic acid, DHA: docosahexaenoic acid, DPA: docosapentaenoic acid, AdA: adrenic acid. Nutrients 2019, 11, 2371 7 of 14 7 of 14 Nutrients 2019, 11, 2371 Increased levels of LA, OA, EPA, AA, DHA, and DPA in DHA-fed mice, and increased levels of LA, OA, EPA, and DHA in GNO-fed mice were observed compared to that in CO-fed mice (Figure 3 and Figure S4). Among the three experiments, an increase in the distribution of AA in GNO-fed mice compared to that of CO-fed mice was observed in two experiments (Figure S4). No signiﬁcant diﬀerence was found in the distribution of AdA between all three groups (Figure 3). Furthermore, maximum fold increase (with respect to the intensity of current ions of each targeted m/z) in the distribution/level of DHA in both GNO-fed and DHA-fed groups was observed compared to those in the CO-fed group (Figure 3 and Figure S5). Compared to that in the CO-fed mice, the distribution of DHA in the brain of SAMP8 mice was increased by 1.85-fold and 3.67-fold in the GNO-fed and DHA-fed mice, respectively (Figure S5). Figure 3. Average intensity (arbitrary unit; a.u.) of DHA and other free fatty acids in sagittal slices of SAMP8 mouse brains. All data are expressed as mean ± SEM. * indicates p < 0.001 compared to CO, and n indicates the number of pixels. Fi 3 A i i ( bi i ) f DHA d h f f id i i l li f Figure 3. Average intensity (arbitrary unit; a.u.) of DHA and other free fatty acids in sagittal slices of SAMP8 mouse brains. All data are expressed as mean ± SEM. * indicates p < 0.001 compared to CO, and n indicates the number of pixels. 3.3. Eﬀects of Dietary Intake of GNO or DHA on Brain Distribution of DHA 3.3. Eﬀects of Dietary Intake of GNO or DHA on Brain Distribution of DHA 3.3. Eﬀects of Dietary Intake of GNO or DHA on Brain Distribution of DHA As the maximum fold changes were obtained in the distribution of DHA following dietary supplementation with GNO or DHA than that obtained with CO supplementation in SAMP8 mouse brains, the distribution of the signal intensity of DHA in the brains of SAMP8 mice (Figure 4A–C) was analyzed. It was found that the distribution of the signal intensity of DHA was not Gaussian (Figure 4B). However, signiﬁcant changes in the spatial distribution of DHA in the brains of GNO-fed or DHA-fed SAMP8 mice compared to that in CO-fed SAMP8 mice (Figure 4A,C and Table S1) were observed. DHA distribution was signiﬁcantly increased in the hippocampus in GNO-fed and DHA-fed mice relative to that in CO-fed mice (Figure 4A,C). Similarly, the dietary intake of both GNO and DHA also signiﬁcantly increased the distribution of DHA in the cerebellum, cerebral cortex, thalamus, hypothalamus, and olfactory bulb in the GNO-fed and DHA-fed mice (Figure 4C, Figure S6, and Table S1). Conversely, distribution of DHA in the septum was increased signiﬁcantly only in DHA-fed mice but not in GNO-fed mice (Figure 4A,C). 8 of 14 Nutrients 2019, 11, 2371 Figure 4. Distribution of DHA in diﬀerent parts of the sagittal sections of SAMP8 mouse brains. CO-fed, GNO-fed, and DHA-fed indicate corn oil-fed SAMP8 mice, green nut oil-fed SAMP8 mice, and DHA-fed SAMP8 mice, respectively. (A) represents H&E stained brain slices and ion images of DHA. (B) represents the distribution of the signal intensity of DHA in SAMP8 mouse brains after supplementation with CO (a), GNO (b), and DHA (c). (C) represents average intensity (a.u.) of DHA in the diﬀerent regions of SAMP8 mouse brains after supplementation with CO, GNO, and DHA. Here * indicates p < 0.001 compared to CO-fed SAMP8 mice. All values are expressed as mean ± SEM. Cb: cerebellum, Cx: cerebral cortex, Hip: hippocampus, OB: olfactory bulb, Tha: thalamus, Hy: Figure 4. Distribution of DHA in diﬀerent parts of the sagittal sections of SAMP8 mouse brains. CO-fed, GNO-fed, and DHA-fed indicate corn oil-fed SAMP8 mice, green nut oil-fed SAMP8 mice, and DHA-fed SAMP8 mice, respectively. (A) represents H&E stained brain slices and ion images of DHA. (B) represents the distribution of the signal intensity of DHA in SAMP8 mouse brains after supplementation with CO (a), GNO (b), and DHA (c). 3.4. Eﬀects of GNO or DHA on Memory Eﬃciency of SAMP8 Mice The results of this investigation indicate that the dietary supplementation of GNO or DHA improved the memory eﬃciency of SAMP8 mice by 1.99-fold and 2.34-fold, respectively, compared to that of the CO-fed SAMP8 mice (Figure 5). Figure 5. Eﬀects of GNO and DHA on memory eﬃciency of SAMP8 mice. All data are presented as mean ± SEM. n indicates the number of mice, and * indicates p < 0.05 compared to CO-fed SAMP8 mice. Figure 5. Eﬀects of GNO and DHA on memory eﬃciency of SAMP8 mice. All data are presented as mean ± SEM. n indicates the number of mice, and * indicates p < 0.05 compared to CO-fed SAMP8 mice. 3.3. Eﬀects of Dietary Intake of GNO or DHA on Brain Distribution of DHA (C) represents average intensity (a.u.) of DHA in the diﬀerent regions of SAMP8 mouse brains after supplementation with CO, GNO, and DHA. Here * indicates p < 0.001 compared to CO-fed SAMP8 mice. All values are expressed as mean ± SEM. Cb: cerebellum, Cx: cerebral cortex, Hip: hippocampus, OB: olfactory bulb, Tha: thalamus, Hy: hypothalamus, ST: septum, n: number of pixels. Spatial distribution of AA in the hippocampus of SAMP8 mice treated with CO, GNO, and DHA was also analyzed. In all three experiments, a signiﬁcant increase in the distribution of AA in the 9 of 14 Nutrients 2019, 11, 2371 hippocampus of GNO-fed and DHA-fed SAMP8 mice was found relative to that in CO-fed SAMP8 mice (Figure S7). hippocampus of GNO-fed and DHA-fed SAMP8 mice was found relative to that in CO-fed SAMP8 mice (Figure S7). 4. Discussion The results of this investigation suggest that the dietary supplementation with GNO or DHA could signiﬁcantly increase the distribution of DHA in the brain of SAMP8 mice. The distribution of several other FFAs associated with dementia was also ameliorated in the SAMP8 mouse brain after GNO or DHA supplementation. To the best of our knowledge, this is the ﬁrst report on the eﬀects of GNO or DHA supplementation on the spatial distribution of DHA and other FFAs in the brain of the animal with dementia. Additionally, the possibility of the potential beneﬁcial eﬀects of GNO or DHA supplementation on memory eﬃciency in dementia using SAMP8 mice was revealed in this investigation. In this study, DESI-IMS was applied in order to examine the spatial distributions of DHA and other FFAs associated with dementia in the brains of male SAMP8 mice. DESI-IMS is a recently developed technique that can be used to analyze the spatial distribution and identification of biomolecules, metabolites, and drugs [28]. Moreover, lipids and FFAs are easily ionized in DESI-IMS, and their metabolism lies at the core of many diseases, including dementia and cancer [4,23,25,32]. Seven molecular ions of FFAs were detected from the mass spectra of DESI-IMS, acquired in negative ion mode from brain slices of SAMP8 mice. Using MS/MS analysis, it was confirmed that AA and DHA corresponded to ions at m/z 303.23 and 327.23, respectively. Based on previous reports and mass accuracy, candidate molecules against the remaining five ions were assigned [30]. Analyzing lipid standards using the same instruments and parameters, which were used to acquire mass spectra from SAMP8 mouse brains, it was also confirmed that all fatty acids were detected as free fatty acids in this study. A decrease in the total amount of LA, OA, EPA, AA, DHA, and DPA in the brains of humans and AD animal models—the most common form of dementia due to changes in the metabolism of FFAs—has been reported in previous studies [2,10,33]. Omega-3 PUFAs enhance fatty acid oxidation, increase ﬂuidity and function of membrane proteins, regulate expression of genes related to signal transmission, serve as precursors to second messengers, and inhibit inﬂammation [5,34–36]. Using DESI-IMS, 10 of 14 Nutrients 2019, 11, 2371 signiﬁcant improvements in the distribution of LA, OA, EPA, AA, DHA, and DPA in the brains of SAMP8 mice after GNO or DHA supplementation were observed compared to CO supplementation. 4. Discussion These results suggest that dietary supplementation with GNO or DHA in SAMP8 mice ameliorated the dementia associated deﬁciency of FFAs. GNO contains about 50% α-linolenic acid (precursor of DHA and EPA), 33% LA (precursor of AA), and 8% OA [3,18]. These fatty acids of GNO may have a role to improve the distribution of FFAs observed in the brain of GNO-fed SAMP8 mice in this study. DHA also can be converted to EPA and DPA through a process known as retroconversion and may help to improve DPA and EPA distribution in the brain of DHA-fed SAMP8 mice [37]. However, there is a paucity of data about the eﬀects of DHA supplementation on the distribution or levels of LA and AA in animals. These FFAs play important physiological roles and can also be converted into other PUFAs and metabolites that are structural components of the cell membrane and crucial for several brain functions [3,10,36]. AA is one of the most abundant FA found in the brain. Although AA plays a notable role in inﬂammation, it acts as a precursor for signaling molecules and as a potent activator of gene transcription factors [1]. It also facilitates neurite overgrowth by stimulating syntaxin 3, which is critical for neuronal development [38]. Among all detected FFAs, the maximum increase in the distribution of DHA was found to occur in the brains of GNO-fed or DHA-fed SAMP8 mice. The distribution of DHA was increased by 1.85-fold and 3.67-fold in the brains of GNO-fed and DHA-fed SAMP8 mice, respectively, when compared to CO-fed SAMP8 mice. ToexaminethedistributionofDHAinthedifferentregionsofSAMP8mousebrains, theregional-specific analysis was performed using images obtained from DESI-IMS for molecular ions of DHA at m/z 327.23. This study demonstrates that the dietary intake of GNO or DHA significantly augmented DHA distribution in most of the regions of SAMP8 mice brain. The accumulation of DHA in the hippocampus, the region where adult neurogenesis occurs and impacts memory function [39,40], in GNO-fed or DHA-fed SAMP8 brains was noted in this study. A decreased level of DHA was found in the hippocampus of SAMP8 mice in a previous study [41]. According to previous reports, DHA supplementation augments brain DHA levels, enhances neurogenesis, and reduces tau hyperphosphorylation, Aβ deposition, neurotransmission, and neuronal apoptosis [12,14,42]. Neurogenesis also occurs in the olfactory bulb and could be enhanced by increased levels of DHA in the olfactory bulb [43,44]. 4. Discussion In this study, the increased distribution of DHA in the olfactory bulb of GNO-fed and DHA-fed SAMP8 mice was found compared to that in the CO-fed group. Additionally, increased levels of DHA in the cerebral cortex, cerebellum, hypothalamus, and thalamus were observed in SAMP8 mouse brains in both DHA-fed and GNO-fed groups compared to that of CO-fed SAMP8 mice. DHA is also expected to improve the functions of these brain regions. The cerebral cortex controls prospective memory and motor function [45], the cerebellum plays a signiﬁcant role in goal-directed behavior and motor control [46,47], the thalamus plays important roles in memory [48], and the hypothalamus controls basic physiological functions, including emotion, reproduction, metabolism, energy expenditure, and ﬂight responses [49]. After DHA, AA is the 2nd FFA in which the most signiﬁcant change was found in DHA-fed and GNO-fed SAMP8 mice compared to CO-fed SAMP8 mice. An increased distribution of AA in the hippocampus of DHA-fed and GNO-fed SAMP8 mice compared to that of CO-fed SAMP8 mice was also observed in this study. LA found in GNO might increase the distribution of AA in the hippocampus of GNO-fed mice. However, to the best of our knowledge, there is a lack of previous reports about the eﬀects of DHA supplementation on the distribution of AA in the animal brain. Further study is required to explore the mechanism by which DHA can aﬀect the distribution of AA in the brain. Dietary intake of DHA improves memory function in both experimental animal models and humans with dementia [50,51]. The recent meta-analysis study also demonstrated that DHA could improve memory function in healthy adults [52]. In order to explore the eﬀects of dietary intake of GNO or DHA in memory eﬃciency, a Y-maze test, which is used to measure short-term memory and stereotypic behavior [53], was performed. It was observed that the memory eﬃciency of SAMP8 mice 11 of 14 11 of 14 Nutrients 2019, 11, 2371 was improved by 1.99-fold and 2.34-fold in GNO-fed and DHA-fed mice, respectively, compared to CO-fed SAMP8 mice. All ﬁndings of this study suggest that both GNO and DHA supplementation improves brain DHA distribution and memory eﬃciency of SAMP8 mice. Currently, the increased demand for DHA containing ﬁsh oil, resulting from its tremendous health beneﬁts, is proving to be detrimental to ﬁsh species and numbers [3]. 5. Conclusions In summary, the dietary intake of GNO or DHA can ameliorate dementia-associated abnormal distribution of DHA in the brain and memory deﬁciency. Furthermore, GNO or DHA supplementation also can increase the distribution of other FFAs in the brain associated with dementia. Therefore, this study suggests the possibility of GNO or DHA supplementation for the prevention of dementia, and the potentiality of GNO as an alternative to ﬁsh oil DHA in the future. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/10/2371/s1, Figure S1: Representative negative ion DESI mass spectrum from sagittal slices of SAMP8 mouse brains and detection of m/z of interest (7 m/z). (A) Full mass spectrum. (B) Expanded view of A. Figure S2: DESI-MS/MS mass spectra of m/z 327.23 (A–D) and 303.23 (E–H) in negative ion mode. Similar fragments were detected in both samples and standards. Figure S3: DESI-IMS mass spectra in negative ion mode from ﬁve lipid standards, OA (A), OA containing LPC (B), OA containing PC (C), DHA (D), and DHA containing PC (E). Figure S4: Average intensity (a.u.) of DHA and other free fatty acids in the sagittal slices of SAMP8 mouse brains from (data from three experiments). FigureS5: Fold changes in the distribution of free fatty acids in the brains of GNO-fed and DHA-fed SAMP8 mice compared to those of CO-fed SAMP8 mice. Figure S6: Distribution of DHA in diﬀerent regions of SAMP8 mouse brains (data from three experiments). Figure S7: Distribution of arachidonic acid (AA) in the hippocampus of SAMP8 mice. Table S1: Fold changes in the distribution of DHA in the diﬀerent brain regions of DHA-fed and GNO-fed SAMP8 mice compared to CO-fed SAMP8 after the supplementation of CO, GNO, and DHA. Author Contributions: Conceptualization, E.T., M.S., and A.I.; Formal analysis, A.I., E.T., N.W., H.T., M.A.M., F.E., and S.S.; Funding acquisition, M.S. and E.T.; Supervision, M.S.; Investigation, M.S., E.T., M.H., I.Y., M.F., and T.K.I.; Methodology, M.S., M.F., S.S., T.S., N.W., H.T., and A.I.; Writing original draft, A.I.; Review and editing the draft copy, M.S., M.H., N.W., H.T., T.K.I., T.S., I.Y., F.E., and M.A.M. 4. Discussion This study suggests the prospective possibility of GNO as a sustainable source of DHA alternative to ﬁsh oil. However, further studies are required to investigate the role and mechanism of increased brain DHA following the dietary supplementation of GNO or DHA to improve memory eﬃciency and other pathological hallmarks of dementia in animal models and humans. 5. Conclusions Funding: This study was supported by Showa Women’s University research grants, MEXT/JSPS KAKENHI Grant Number JP15H05898B1, Japan Agency for Medical Research and Development (AMED) under Grant Number JP19gm0910004, Japan Science and Technology Agency (JST) Program for Creating STart-ups from Advanced Research and Technology (START) under Grant Number ST292006UT, and Imaging Platform supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Acknowledgments: We are grateful to Tsuyoshi Watanabe for providing DHA (Tama Biochemical Co., Ltd.) and Norihisa Oohashi for providing green nut oil (NPO Corporation Arcoiris). 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https://openalex.org/W4241574630	https://www.nature.com/articles/s41598-020-74328-x.pdf	English	null	Lack of Tocilizumab effect on mortality in COVID19 patients	Research Square (Research Square)	2,020	cc-by	3,550	Lack of tocilizumab effect on mortality in COVID19 patients OPEN Gregory E. Holt, Mayank Batra , Mukunthan Murthi, Shweta Kambali, Kayo Santos, Maria Virginia Perez Bastidas, Huda Asif, Sara Haddadi, Sixto Arias & Mehdi Mirsaeidi * Off-label tocilizumab use in COVID-19 patients reflects concern for cytokine release syndrome. Comparison of matched COVID-19 pneumonia patients found elevated IL-6 levels correlated with mortality that did not change with tocilizumab administration. Correlating mortality with increased IL-6 doesn’t imply causality however lack of improvement by tocilizumab requires further clinical trial alterations. The lack of proven medications for COVID-19 beget the widespread use of off label therapeutics based on theo- retical efficacy. Early data correlated elevated levels of IL-6 with increased mortality1. This finding led clinicians to use the humanized anti-IL6 receptor antagonist, tocilizumab, to attenuate IL-6 levels in infected patients based on its proven benefit in patients suffering from cytokine release syndrome after chimeric antigen receptors (CAR)-T cell infusion2,3. In COVID-19, the question of whether elevated IL-6 levels reflect an overwhelming viral infection or directly cause immunopathology responsible for a patient’s poor outcome remains unanswered. We present our experience with tocilizumab in conjunction with the published literature to argue for the urgent need to understand a disease before carte blanche application of unproven therapies in single arm trials. We aimed to find the effect of tocilizumab on mortality of COVID19 in a well-matched population cohort study. if y p p y We performed a retrospective cohort study of matched patients admitted to the University of Miami Hospital with a confirmed diagnosis of COVID-19 approved by University of Miami. i g pp y y Institutional Review Board (IRB #20,200,441). All procedures in this study were performed in accordance with relevant guidelines and regulations. The IRB waived a requirement for informed consent in the context of minimal risk research.t Tocilizumab was administered to patients after consultation with an Infectious Disease driven committee for COVID19 who required oxygen ≥ 4 L per minute via nasal cannula and had specific levels of at least 4 bio- markers; IL-6 > 40 pg/mL, CRP > 10 mg/dL, D- dimer > 1 mcg/mL FEU, ferritin > 1,000 ng/mL, or LDH > 350 units. Out of 250 confirmed COVID-19 patients, 32 (12.5%) received Tocilizumab during hospitalization and enrolled in the study. Patients received 400 mg tocilizumab as a single intravenous infusion. 24 patients received tocilizumab when started mechanically ventilation and rest on supplemental oxygen. www.nature.com/scientificreports www.nature.com/scientificreports Division of Pulmonary and Critical Care, University of Miami, Miami, FL, USA. email: msm249@med.miami.edu Scientific Reports \| (2020) 10:17100 Lack of tocilizumab effect on mortality in COVID19 patients OPEN Thirty patients who did not receive tocilizumab were matched for gender, age, ICU admission, and qSOFA score and included in the control group. Demographic, clinical, laboratory data, and in-hospital mortality data were collected from the medical records. For descriptive analysis of ordinal variables, we used Mantel–Haenszel methods. Continuous variables were reported as median and interquartile range (IQR). Medians of two groups were tested using Wilcoxon-Mann–Whitney test. We used Cox’s proportional hazards model to analyze survival time and multi- variate analysis to test the effect of each independent variable on mortality. A p value less than 0.05 was considered statistically significant.h y gi Nineteen patients (30.6%) died during this study with 14 (22.6%) dying in the ICU. The majority of deaths occurred in subjects older than 75 years (OR 7.1, p = 0.049). Subjects with IL-6 levels over 580 pg/mL had an increased mortality (OR 54.7, p = 0.007) (Table 1). In multivariate analysis, tocilizumab administration had no discernible effect on mortality (OR 0.3, p value 0.36). Additional variables that were conclusive using the uni- variate analysis but did not hold up in multivariate analysis include; residents of nursing homes/long term care facility (OR 13.0, p = 0.12), diabetes mellitus (OR1.5, p = 0.70) solid tumors (OR 3.70, p = 0.57), ferritin ≥ 1631 (OR 8.3, p = 0.12), and altered mental status (OR 0.9, p = 0.95) (Fig. 1). Figure 2 shows survival analysis result and showing no significant difference in mortality between subjects treated and untreated with tocilizumab. g gif y j Our data show survivors had mean IL-6 levels of 177.9 + /- 227.9 whereas non-survivors had mean levels of 1384.2 + /- 1234.7 congruent with reports that elevated IL-6 levels are associated with poor outcomes in patients with COVID19 viral pneumonia4,5. The literature showing this correlation predominantly only measures admis- sion IL-6 levels (most < 50 pg/mL) but does not follow them throughout the clinical course5. A recent study in Chest followed IL-6 levels after tocilizumab administration and found an immediate increase followed by a steady decrease but did not differentiate IL-6 levels between survivors and non-survivors4. Lack of tocilizumab effect on mortality in COVID19 patients OPEN Data from China \| https://doi.org/10.1038/s41598-020-74328-x Scientific Reports \| (2020) 10:17100 www.nature.com/scientificreports/ Demographics (N = 62) Survived N (%) Did not survive N (%) p value Age, Median (IQR) year 62 (42–82) 75 (58–92) 0.006 Age ≥ 75 (N = 15) 5 (11.6) 10 (52.6) 0.001 Male (N = 44) 30 (69.8) 14 (73.7) 0.754 Hispanics (N = 34) 23 (56.1) 11 (64.7) 0.545 White (N = 33) 20 (51.3) 13 (76.5) 0.085 Black (N = 15) 13 (33.3) 2 (11.8) 0.109 Asian (N = 2) 1 (2.6) 1 (5.9) 0.55 More than one race (N = 6) 5 (12.8) 1 (5.9) 0.452 Tobacco cigarette use (N = 39) 27 (62.8 12 (63.2) 0.978 Vaping (N = 51) 37 (86) 14 (73.7) 0.247 Alcohol (N = 50) 35 (81.4) 15 (78.9) 0.822 Marijuana (N = 49) 32 (74.4) 17 (89.5) 0.194 Symptoms at or after 48 h of hospital admission Temperature > = 100°F (N = 26) 18 (41.9) 8 (42.1) 0.986 Cough (N = 44) 32 (76.2) 12 (66.7) 0.447 Sore throat (N = 2) 1 (2.4) 1 (5.6) 0.553 Rhinorrhea (N = 4) 3 (7.3) 1 (6.3) 0.887 Dyspnea (N = 43) 30 (69.8) 13 (68.4) 0.916 Fever (N = 42) 29 (67.4) 13 (72.2) 0.713 Chills (N = 21) 13 (31) 8 (44.4) 0.318 Myalgias (N = 16) 10 (24.4) 6 (33.3) 0.478 Abdominal pain (N = 5) 4 (9.8) 1 (5.9) 0.636 Diarrhea (N = 5) 3 (7.3) 2 (11.1) 0.632 Nausea/vomiting (N = 7) 5 (12.2) 2 (11.8) 0.963 Altered mental status (N = 8) 3 (7.1 5 (29.4) 0.035 Pre-admission oxygen use (N = 5) 1 (2.4) 4 (21.1) 0.039 Inhaled steroid use (N = 6) 4 (9.5) 2 (11.1) 0.851 qSOFA, Median (IQR) 0.001 (0–0.5) 1 (0–2) 0.582 Medications prior to hospital admission Prednisone 6 (14.3) 2 (11.1) 0.741 ACE inhibitors 4 (9.5) 3 (17.6) 0.389 Angiotensin Receptor Blockers 7 (16.7) 3 (17.6) 0.928 Statins 10 (23.8) 7 (41.2) 0.187 Emergency Room visit within 12 months 10 (24.4) 4 (22.2) 0.857 Hospital admission within last 12 months 11 (26.8) 5 (27.8) 0.94 Nursing home/long term care facility residents 5 (11.9) 9 (47.4) 0.004 Findings at Chest X-Ray or CT images obtained while in hospital Ground glass opacities 9 (22.0) 5 (26.3) 0.71 Consolidations 10 (24.4) 8 (42.1) 0.168 Pleural effusions 5 (12.2) 5 (26.3) 0.181 Bilateral infiltrates 27 (65.9) 19 (31.7) 0.552 Comorbidities Chronic Obstructive Pulmonary Disease 4 (9.5) 1 (5.3) 0.58 Supportive oxygen before admission 16 (42.1 13 (68.4) 0.066 Congestive Heart Failure 1 (2.4) 2 (10.5) 0.211 Atrial fibrillation 4 (9.5) 2 (10.5) 0.903 Hypertension 23 (54.8) 14 (73.7) 0.167 Stroke 4 (9.5) 2 (10.5) 0.903 Dementia 2 (4.8) 1 (5.3) 0.933 Chronic Renal Failure 2 (4.8) 1 (5.3) 0.933 Diabetes Mellitus 10 (23.8) 13 (68.4) 0.02 Lymphoma 2 (4.8) 1 (5.3) 0.933 Solid tumor 1 (2.4) 4 (21.1) 0.039 Laboratory findings at or within 48 h of admission to hospital Procalcitonin ≥ 1.15 ng/mL 7 (16.3 5 (26.3) 0.36 Fibrinogen ≥ 649 mg/dL 2 (4.7) 1 (5.3) 0.918 Continued https://doi.org/10.1038/s41598-020-74328-x Scientific Reports \| (2020) 10:17100 \| www.nature.com/scientificreports/ Demographics (N = 62) Survived N (%) Did not survive N (%) p value IL-6 > 580 pg/mL 1 (2.3) 4 (21.1) 0.037 IL-6 Median (IQR) pg/mL 81.8 (0–264.9) 1197.3 (0–3738.9) 0.097 Ferritin > 1631 ng/mL 6 (14.0) 7 (36.8) 0.048 C-Reactive Protein ≥ 21 7 (16.3) 6 (31.6) 0.179 Mechanical ventilation use 21 (65.6) 15 (83.3) 0.19 Positive blood culture obtained after tocilizumab 5 (16.1) 2 (11.8) 0.683 Medications for COVID19 Tocilizumab 22 (51.2) 10 (52.6) 0.915 Tocilizumab administration post-admission, Median (IQR) day 2 (0–5) 2 (0–6) 0.703 Tocilizumab for more than 4 days 6 (14.0) 2 (10.5) 0.711 Chloroquine/Hydroxychloroquine 31 (72.1 15 (32.6) 0.571 Macrolides 33 (76.7) 16 (84.2) 0.508 Steroids 26 (60.5) 10 (52.6) 0.565 Outcome Hospital stay duration, Median (IQR) day 22 (0–47) 11 (0–30) 0.102 ICU stay duration, Median (IQR) day 5 (0–24) 7 (0–21) 0.582 Patient delay, Median (IQR) day 4 (0–10) 4 (0–10) 0.703 Physician delay**, mean (SD) day 3 (0–10) 4.0 (0–11) 0.609 Readmission 3 (7.0) 1 (5.3) 0.801 ICU admission 29 (67.4) 17 (89.5) 0.083 bl h d l l h d f b h Table 1. Lack of tocilizumab effect on mortality in COVID19 patients OPEN Forest plot showing the variables used in the multivariate model among COVID19 subjects. Hosm lameshow score > 0.05, IL6 and Ferritin units: pg/ml. supraphysiologic IL-6 levels in CAR-T infusion both correlate with high tumor burdens17 and appear to be directly responsible for disease as administration of tocilizumab dramatically improves clinical status usually within 48 h3. Our data and another retrospective cohort trial of matched patients showed tocilizumab had no effect on mortality18. supraphysiologic IL-6 levels in CAR-T infusion both correlate with high tumor burdens17 and appear to be directly responsible for disease as administration of tocilizumab dramatically improves clinical status usually within 48 h3. Our data and another retrospective cohort trial of matched patients showed tocilizumab had no effect on mortality18.h f y There are several limitations to this retrospective cohort study including small sample size and retrospective cohort design. Tocilizumab was given based on clinical parameters and biomarkers assumed to indicate IL-6 mediated immunopathology. Although IL-6 is correlated with poorer outcomes, in COVID19 we do not know if, at what level or at what time point IL-6 leads to immunopathology. Tocilizumab may have failed to influence mortality because IL-6 may not be either responsible for, or the only cytokine involved in immunopathology. If IL-6 is responsible for CRS in COVID-19, it is unknown at what level or time point it changes from having anti- viral properties to causing immunopathology. Use of tocilizumab in patients regardless of IL-6 levels may have diluted out patients for whom tocilizumab may have benefited obscuring its effect on mortality. Tocilizumab would not help patients who did not produce pathologic IL-6 levels but could be detrimental if lower IL-6 levels were necessary to fight the viral infection15. yi g We believe the need to “do something” has superseded the need to evaluate disease to apply clinical trials based on data. We argue that evaluation of immune parameters in COVID19 patients need to first be studied to ensure that IL-6 is involved in immunopathology and second to determine at what level or time point in the clinical course of infection, IL-6 produces immunopathology. Serial measurements of key cytokines in COVID19 may characterize IL-6 and additional cytokine levels to correlate with clinical outcomes, before administration of tocilizumab. Received: 8 July 2020; Accepted: 23 September 2020 Received: 8 July 2020; Accepted: 23 September 2020 Lack of tocilizumab effect on mortality in COVID19 patients OPEN Demographic and clinical characteristics, and outcomes of subjects with COVID19. Table 1. Demographic and clinical characteristics, and outcomes of subjects with COVID19. Figure 1. Cox regression showing no statistical difference in mortality between COVID 19 subjects treated and untreated tocilizumab therapy. Blue: shows treated with Tocilizuman, Black: shows untreated with Tocilizumab. Variables in the model: Age > = 75, IL6 > = 580, and Tocilizumab. p value, HR (95%CI) for Tocilizumab: 0.75, 0.9 (0.3–2.2). Figure 1. Cox regression showing no statistical difference in mortality between COVID 19 subjects treated and untreated tocilizumab therapy. Blue: shows treated with Tocilizuman, Black: shows untreated with Tocilizumab. Variables in the model: Age > = 75, IL6 > = 580, and Tocilizumab. p value, HR (95%CI) for Tocilizumab: 0.75, 0.9 (0.3–2.2). found patients with rising IL-6 levels greater than 4000 pg/mL perished despite receiving tocilizumab6. These IL-6 levels begin to reach those seen in cytokine release syndrome (CRS) following CAR T cell infusion where levels reach a median value of 8,309 pg/mL for grade 4 or 5 CRS7.hi The finding of an elevated cytokine level in disease does not prove causality regardless of the degree of cor- relation. However, rapid reversal of a clinical syndrome through use of cytokine specific blockade provides good data linking that cytokine to disease pathogenesis. Use of tocilizumab for CRS in patients treated with CAR T cells causes a dramatic improvement in disease usually within 48 h8,9. To date, tocilizumab has not had the same dramatic effect on reversing COVID-19 pneumonia like it does in CRS from CAR-T cell infusion despite papers suggesting possible benefit when compared to historical controls10–14.t gg g pi p Comparison of IL-6′s role in viral infections versus CRS after CAR-T cell infusion is informative. IL-6 is an important cytokine responsible for promoting anti-viral T cell responses, inhibiting viral replication and resolving inflammation to promote tissue repair15. However, mouse models of viral infections that artificially create supraphysiologic IL-6 levels (10,000 pg/mL) show viral persistence and increased immunopathology16. Immune stimulating agents in cancer, e.g. CAR T cells, are efficacious partly because they are impervious to negative immune regulation and allow unfettered immune reactions against tumor cells. Not surprisingly, the Scientific Reports \| (2020) 10:17100 \| https://doi.org/10.1038/s41598-020-74328-x www.nature.com/scientificreports/ Figure 2. Forest plot showing the variables used in the multivariate model among COVID19 subjects. Hosmer lameshow score > 0.05, IL6 and Ferritin units: pg/ml. Figure 2. Lack of tocilizumab effect on mortality in COVID19 patients OPEN After measuring the levels of cytokines in the clinical course of a COVID19 infection, trials should be attempted to apply immune modulating therapy to patients for whom the immune system is causing disease via dysregulation. References 1. Ruan, Q., Yang, K., Wang, W., Jiang, L. & Song, J. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intens. Care Med. 46, 846–848. https://doi.org/10.1007/s00134-020-05991-x (2020). 2. Kewan, T. et al. Tocilizumab for treatment of patients with severe COVID-19: a retrospective cohort study. EClinicalMedicine 24, 100418. https://doi.org/10.1016/j.eclinm.2020.100418 (2020). Acknowledgmentsh g The authors would like to thank the physicians and nurses of the COVID-19 units of University of Miami and all of the patients for their participation. g The authors would like to thank the physicians and nurses of the COVID-19 units of University of Miami and all of the patients for their participation. www.nature.com/scientificreports/ Kinetics and biomarkers of severe cytokine release syndrome after CD19 chimeric antigen receptor-modified T-cell therapy. Blood 130, 2295–2306. https://doi.org/10.1182/blood-2017-06-793141 (2017).fi py p g ( ) 18. Campochiaro, C. et al. Efficacy and safety of tocilizumab in severe COVID-19 patients: a single-centre retrospective cohort study. Eur. J. Intern. Med. 76, 43–49. https://doi.org/10.1016/j.ejim.2020.05.021 (2020). py p g ( ) 18. Campochiaro, C. et al. Efficacy and safety of tocilizumab in severe COVID-19 patients: a single-centre retrospective cohort study. Eur. J. Intern. 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W2146112440.txt	https://www.frontiersin.org/articles/10.3389/fgene.2012.00115/pdf	en		The Choice between MapMan and Gene Ontology for Automated Gene Function Prediction in Plant Science	Frontiers in genetics	2,012	cc-by	9,981	ORIGINAL RESEARCH ARTICLE published: 28 June 2012 doi: 10.3389/fgene.2012.00115 The choice between MapMan and Gene Ontology for automated gene function prediction in plant science Sebastian Klie 1 and Zoran Nikoloski 2 * 1 2 Genes and Small Molecules Group, Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany Systems Biology and Mathematical Modeling Group, Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany Edited by: John Hancock, Medical Research Council, UK Reviewed by: John Hancock, Medical Research Council, UK Pankaj Jaiswal, Oregon State University, USA Keiichi Mochida, RIKEN, Japan *Correspondence: Zoran Nikoloski , Systems Biology and Mathematical Modeling Group, Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm D-14476, Germany. e-mail: nikoloski@ mpimp-golm.mpg.de Since the introduction of the Gene Ontology (GO), the analysis of high-throughput data has become tightly coupled with the use of ontologies to establish associations between knowledge and data in an automated fashion. Ontologies provide a systematic description of knowledge by a controlled vocabulary of deﬁned structure in which ontological concepts are connected by pre-deﬁned relationships. In plant science, MapMan and GO offer two alternatives for ontology-driven analyses. Unlike GO, initially developed to characterize microbial systems, MapMan was speciﬁcally designed to cover plant-speciﬁc pathways and processes. While the dependencies between concepts in MapMan are modeled as a tree, in GO these are captured in a directed acyclic graph. Therefore, the difference in ontologies may cause discrepancies in data reduction, visualization, and hypothesis generation. Here provide the ﬁrst systematic comparative analysis of GO and MapMan for the case of the model plant species Arabidopsis thaliana (Arabidopsis) with respect to their structural properties and difference in distributions of information content. In addition, we investigate the effect of the two ontologies on the speciﬁcity and sensitivity of automated gene function prediction via the coupling of co-expression networks and the guilt-by-association principle. Automated gene function prediction is particularly needed for the model plant Arabidopsis in which only half of genes have been functionally annotated based on sequence similarity to known genes. The results highlight the need for structured representation of speciesspeciﬁc biological knowledge, and warrants caution in the design principles employed in future ontologies. Keywords: Arabidopsis thaliana, design principles of ontologies, gene function prediction, Gene Ontology, information content, MapMan INTRODUCTION With the ever increasing availability and quality of highthroughput data from all levels of cellular organization (e.g., transcriptome, proteome, and metabolome), ontologies have become an integral part of multivariate data analysis to facilitate biological interpretations. Accumulated knowledge in biology, unlike other scientiﬁc ﬁelds, is rather difﬁcult to capture, and convey with mathematical formalisms. Nevertheless, ontologies offer the means for structured representation of knowledge gathered in various (electronic) written forms (e.g., text books, journal articles, databases), whereby the structure pertains to the relationships between knowledge concepts. Since ontologies are intended to represent corpora of knowledge, often in a particular ﬁeld, the considered concepts can be used to annotate entities from the ﬁeld of research. Decade-long research efforts in this area, including annotation schemes such as the MIPS functional categories as well as the KEGG ontology (Ruepp et al., 2004), have resulted in ontologies tailored to different aspects of biological research, from genes and pathways to species-speciﬁc tissues, organs, and entire anatomies (Bard and Rhee, 2004). Two aspects of using biological ontologies have already been adequately addressed and thoroughly investigated, namely: (1) statistical tests for enrichment of www.frontiersin.org ontological concepts (Rivals et al., 2007), (2) categorization and choice of semantic similarity measures for comparison of ontological concepts (Guzzi et al., 2011). However, the integration of biological ontologies, to facilitate interoperability of genomic databases, and their comparison, with the aim of selecting suitable ontologies, can still be regarded as pressing issues in bioinformatics and computational biology (Stein, 2003; Punta and Ofran, 2008). In combination with methods from multivariate data analysis (e.g., clustering and separation), structured biological knowledge allows for automated reasoning and statistically sound inferences in biology. This is particularly relevant due to the recent surge of methods and applications in network-driven co-expression analysis of transcriptomics (i.e., gene expression) data. Co-expression networks provide the medium for transfer of gene annotation following the guilt-by-association (GBA) principle, whereby known (and enriched) function in a set of genes is propagated to the genes of unknown function in the set. Solutions for automated gene function annotation are still relevant even for well-investigated model organisms, such as Arabidopsis thaliana (Arabidopsis) with ∼27,000 genes of which only half have been functionally annotated based on sequence similarity to known genes, while the function June 2012 \| Volume 3 \| Article 115 \| 1 Klie and Nikoloski of mere 13% has been experimentally conﬁrmed (Lamesch et al., 2012). In modern plant biology, there are two widely used ontologies: the Gene Ontology (GO) and MapMan. While the general GO has originated as species-unspeciﬁc, MapMan was initially speciﬁcally tailored to Arabidopsis. Furthermore, the latter has been extended to cover other plants such as maize (Doehlemann et al., 2008), Medicago (Tellström et al., 2007), tomato (Urbanczyk-Wochniak et al., 2006), and potato (Rotter et al., 2007). With respect to the nomenclature of concepts, the MapMan ontology comprises a set of 34 tree-structured bins, describing the central metabolism as well as other cellular processes (e.g., stress responses). On the other hand, GO is a collection of concepts, called terms, which are connected via is a and part of relations aimed at functionally categorizing genes (for details of scope and structure of GO, the reader is directed to, Ashburner, 2000; Stevens et al., 2000; Blake and Harris, 2002). Moreover, GO can be regarded as a collection of three ontologies that correspond to independent categories of gene function: molecular function (GO-MF), biological processes (GOBP), and cellular component (GO-CC). Functional categorization of genes can also be performed across species with the help of high-level GO terms, reducing GO to the so-called GO slim ontology. Besides the generic species-unspeciﬁc version, there are GO slim ontologies which are designed for speciﬁc species, e.g., Saccharomyces cerevisiae (Cherry et al., 2012), Arabidopsis (Lamesch et al., 2012), and Drosophila (Adams et al., 2000). In MapMan, the original assignment of bins was based on publicly available gene annotation in TIGR (The Institute for Genomic Research), adopting a process alternating between automatic recruitment, and manual correction (Thimm et al., 2004). Although the two ontologies have both been used in plant research, systematic comparison of GO and MapMan has not yet been undertaken. Assessing the advantages and drawbacks of the two is crucial for the selection of the ontology suitable for automated gene function annotation. Here we present the ﬁndings from the comparative analysis of GO and MapMan, ﬁrst by analyzing similarities and differences with respect to the (1) overall structure and size, and (2) design principles. Here, we suggest suitable preprocessing strategies to alleviate the problem of inconsistent mappings regarding the inheritance of concepts given by the respective structure of the ontology. Furthermore, for the speciﬁc case of the gene annotation for Arabidopsis, we investigate (3) the coverage and (4) biological relevance of concepts within the two ontologies. In addition, we analyze the effect of a particular ontology on the function transfer across genes based on the coupling between the GBA principle and co-expression networks. The ﬁndings from our comparative analysis point out that the domain in which ontologies are used may have a profound effect on the selection of a best-performing alternative. Therefore, our results pinpoint the need for development of methods for objective, systematic, and problem-speciﬁc comparison of biological ontologies as well as formal frameworks for transfer of ontologies in cross-species analyses. RESULTS THE STRUCTURE OF MAPMAN AND GO Although the relationships between two ontological terms in GO and MapMan can be described by is a and part of relationships, the Frontiers in Genetics \| Bioinformatics and Computational Biology Comparison of MapMan and GO structures of the two ontologies differ. While all three categories of GO are structured in the form of a directed acyclic graph (DAG; Yon Rhee et al., 2008), the relationships in MapMan are modeled following a tree structure (cf. Figure 1). The implication of using a DAG as an underlying structure of the ontology is that child concepts may have more than one parent. The multiplicity of parent concepts can be regarded as an advantage, as it provides a high degree of ﬂexibility and may enable powerful grouping, searching, and analysis of genes (Yon Rhee et al., 2008). In contrast, although the tree structure closely resembles the intuitive connotation of a hierarchy of concepts, it sacriﬁces a part of the ﬂexibility when the ontology is updated (e.g., by adding new concepts). A disadvantage of the DAG structure, compared to a tree, is that the depth of a concept cannot be unambiguously deﬁned, since there may exist multiple paths to the root node. Therefore, we deﬁne the depth of a concept in GO (i.e., term) as the shortest path to the root node, corresponding to the minimum concept depth (see Guzzi et al., 2011) for other similar measures). In addition, multiple parent concepts increase the overall number of possible ancestors at the same concept depth. This is particularly the case when comparing the DAG structure of GO with the tree structure of MapMan. Furthermore, the number of potential parent concepts as well as the overall size of an ontology renders it difﬁcult to visualize concept associations for large-scale transcriptomic analyses (for the plethora of available visualization methods see, e.g., Zeeberg et al., 2003; Tsiaras et al., 2008; Carbon et al., 2009; and has effect on statistical hypothesis testing, e.g., in multiple testing scenarios Goeman and Mansmann, 2008). An immediate solution represents GO slim, which categorizes genes on the basis of a relatively small set of high-level GO terms. Like in the tree structure of MapMan, the smaller number of (parent-) terms of the slim ontologies facilitates the interpretability of obtained results. However, similarly to the previous arguments, a small number of parent terms can also turn out to be a disadvantage, as it may lead to a comparatively ﬂatter hierarchy structure, regardless of the actual size of the used ontology. Subsequently, a ﬂat hierarchy may compromise the speciﬁcity and biological relevance of individual concepts due to its coarseness. DESIGN PRINCIPLES OF ONTOLOGIES – CAPTURING BIOLOGICAL CONCEPTS An important characteristic of GO is the division in three nonoverlapping domains of molecular biology–biological process (GO-BP), molecular function (GO-MF), and cellular component (GO-CC; Ashburner, 2000; Harris and Gene Ontology, 2004). While terms in GO-BP domain describe biological objectives and processes in which the annotated genes participate, terms in GOMF characterize biochemical activities that ultimately contribute to biological processes. Finally, GO-CC summarizes the subcellular localization where a gene product is active. In contrast, while MapMan does not have a structure composed of independent categories, one can still distinguish between high- and low-level bins. Since the design principle of MapMan was to intuitively characterize and visualize metabolic pathways and processes (Thimm et al., 2004), high-level bins tend to be similar to terms in the GO-BP ontology, whereas low-level bins often resemble terms from the GO-MF ontology. June 2012 \| Volume 3 \| Article 115 \| 2 Klie and Nikoloski Comparison of MapMan and GO FIGURE 1 \| Comparison of the concepts used in the annotation of the gene coding for naphthoate synthase (at1g60550, 264920_at) in the DAG and tree structure of GO DAG and MapMan, respectively. To illustrate this claim based on the whole annotation of gene products rather than examples of individual concepts, we quantiﬁed the similarity of MapMan bins and GO terms by utilizing a network-based approach. For the purpose of this analysis, nodes correspond to concepts, i.e., terms in GO and bins in MapMan. An edge between two nodes is established if the set of genes which are annotated with the respective terms corresponding to the nodes are similar (cf. Materials and Methods). Figure 2 shows the resulting network which consists of all GOMF and GO-BP terms that exhibit a similarity to at least one MapMan bin. The edges of the resulting network can further be divided by the type of association they model, namely: similarity between MapMan bin and GO-BP term, MapMan bin and GO-MF term as well MapMan bin, and both GO-MF and GO-BP terms. Inspection of the three types of edges in this concept-association network shows that high-level MapMan bins are often associated with terms originating from GO-BP. In contrast, MapMan bins deeper in the hierarchy are predominantly associated with GO-MF terms. A statistical analysis quantiﬁes this observation as the difference of average depth of concepts for the ﬁrst two of the groups of edges is statistically signiﬁcant at the 5% level (Wilcoxon-RankSum test, p-value = 0.016, cf. Figure 3). Here and in the following, we only use the terms from the two GO ontologies, namely: GOMF and GO-BP, since there is no correspondence between GO-CC and any bin in MapMan. GENE ANNOTATION COVERAGE – THE STATUS QUO FOR ARABIDOPSIS The genome of Arabidopsis contains 27,416 protein coding genes according to the latest genome annotation version (TAIR10, November 2010)1 which excludes pseudo genes and genes encoded by transposable elements (Lamesch et al., 2012). Inspection of these mappings shows that a total of 15,238 gene products are 1 http://arabidopsis.org www.frontiersin.org annotated with MapMan bins, while 12,225 and 13,157 genes are annotated by GO-BP and GO-MF terms, respectively. By combining the available annotation of all three ontologies ∼63% of Arabidopsis’ genes can be annotated. The number of genes that are annotated with both MapMan and GO terms (either GO-BP or GO-MF) is ∼87% of the total number of annotated genes with concepts from any of the three ontologies (cf. Figure 4). Furthermore, each ontology contains concepts used in the annotation of a unique set of genes: the contribution of MapMan is slightly larger, with 2,557 unique bins, compared to 625 and 572 terms for GO-MF and GO-BP, respectively (Figure 4). In summary, the coverage of the two ontologies is comparable, which further serves as a justiﬁcation for the undertaken comparative analysis. In addition, we ﬁnd that 3,598 unique GO-BP terms are used to annotate ∼45% of Arabidopsis’ genes. GO-MF contains 2,148 unique terms covering ∼48% of the genes. Finally, 1,361 unique bins of MapMan are used in annotating 56% of Arabidopsis’ genes. Similarly to the overall size of the ontologies, we demonstrate that the average number of parent terms per gene in MapMan is 3 in comparison to 20 and 7 in GO-BP and GO-MF, respectively. Clearly, MapMan is the smaller ontology with roughly one-third of the size of GO-BP. Furthermore, to see whether a comparatively low number of parent terms ultimately results in an overall ﬂatter hierarchy structure in the case of MapMan, we analyze the differences in the distribution of depth in the two ontologies. Again, we contrasted the concept depth distribution on the current state of ontological gene annotation in Arabidopsis. Here, for each annotated gene, we determined the depth of every associated term and all of its parents (further deﬁned as “complete ontology”, see Materials and Methods). As shown in Figure 5, MapMan indeed represents a ﬂatter hierarchy: while both term depth distributions of the two GO categories closely resemble a normal distribution with a mean ∼ = median term depth of ∼5 (sample skewness: June 2012 \| Volume 3 \| Article 115 \| 3 Klie and Nikoloski FIGURE 2 \| Network of associations between MapMan bins and GO terms. Blue nodes represent MapMan bins, yellow denote GO-BP terms, and orange nodes correspond to GO-MF terms. The size of the nodes representing MapMan bins corresponds to their depth in the ontology. Note that the low-level bins (small blue nodes) correspond to terms in GO-MF, GO-MF = 0.01,GO-BP = 0.26), the term depth distribution of MapMan is skewed toward lower values with median term depth of three (sample skewness: 0.69). In addition, the maximum term depth is lower, and is of value seven in MapMan and 10 in both GO categories, respectively. INFORMATION CONTENT OF ONTOLOGICAL TERMS Common to both ontologies is that high-level concepts describe general processes, functions, or structures, while low-level concepts are more speciﬁc. The previous claim that MapMan constitutes a ﬂatter hierarchy structure, compared to GO, needs further investigation to ascertain whether the structure of MapMan can be used equally well in elucidating biologically meaningful information from its ontological concepts (i.e., bins). Here we rely on the information content (IC) of an ontology concept to quantify its speciﬁcity by accounting for the overall number of genes annotated with it (Resnik, 1995). Brieﬂy, the information content of an ontology concept is lower as its speciﬁcity decreases; the more abstract a concept, or broader an ontological category, the lower its information content (see Material and Methods). Figure 6 shows a histogram of the IC of all MapMan, GO-MF and GO-BP used in the annotation of the Arabidopsis’ genome. One can observe that both GO ontologies exhibit a higher maximum IC as well as more terms of large IC. Moreover, the median IC of 9.23 for MapMan is smaller than that of GO ontologies, i.e., 10.4 for GO-MF and 10.82 Frontiers in Genetics \| Bioinformatics and Computational Biology Comparison of MapMan and GO while high-level bins (large blue nodes) are associated to terms in GO-BP. The edges of the network are divided by the type of association they model, namely: association between MapMan bin and GO-BP term (left), MapMan bin and GO-MF term (middle) as well MapMan bin and both GO-MF and GO-BP terms (right). for GO-BP. This implies a slightly coarser grouping of processes and functions in the case of MapMan. However, one can also observe that MapMan contains more terms of average IC (∼5.5 to ∼8.5). Besides the analysis of the distribution of ICs for concepts of an ontology, it is important to also investigate the interplay between the underlying structure (captured by the concept depth) and IC to characterize the level at which a deeper hierarchy relates to more speciﬁc sets of genes. This dependence between concept depth and IC is visualized in Figure 7 with the help of box plots. One can observe that all three ontologies exhibit an asymptotic trend of the median IC values per concept depth. Interestingly, none of the ontologies displays a gradual trend of a linearly increasing IC with the increasing concept depth. Further, this non-linear behavior can be modeled using classical MichaelisMenten kinetics (Lehninger et al., 2008), which relates the rate of a reaction (dependent variable) with the (saturating) concentration of its substrate (independent variable). The relation is fully described by two parameters: V max , representing the maximum rate achieved at maximum (saturating) substrate concentrations, and K m , denoting the substrate concentration at which the rate is half of V max . Analogously to this classical enzyme kinetics, we take V max to denote maximum IC achieved at maximum concept depth and K m , the concept depth at which the IC is V max /2. By using non-linear (least-squares) regression (Leskovac, 2003), we June 2012 \| Volume 3 \| Article 115 \| 4 Klie and Nikoloski Comparison of MapMan and GO GO-MF and GO-BP, respectively. Therefore, we conclude that, in the case of Arabidopsis, all three ontologies possess the similar structural capabilities to allow for an adequate biologically meaningful discrimination of concepts and genes. EMPLOYING MAPMAN AND GO FOR AUTOMATED GENE FUNCTION ANNOTATION – THE CASE STUDY OF ARABIDOPSIS FIGURE 3 \| Concept depth of MapMan bins associated with GO terms based on similarity of the annotated genes (cf. Figure 2). Low-level MapMan bins tend to represent terms from the GO-MF ontology (left boxplot), while high-level MapMan bins are associated with terms from GO-BP (middle). The difference of the bin depth for these two groups is statistically signiﬁcant at level α = 0.05. FIGURE 4 \| Venn diagram illustrating the overlap in the gene annotation of Arabidopsis thaliana with concepts from MapMan, GO-BP, and GO-MF, respectively. obtain estimates for the constants V max and K m (cf. Materials and Methods). Interestingly, we ﬁnd the all determined K m values are close to ∼1, relating to V max /2 of 6.08, 6.04 and 6.76 for MapMan, www.frontiersin.org The current incompleteness of available gene annotation for Arabidopsis clearly emphasizes the need for automated gene function prediction, even in the case of well-studied model organism. In addition to sequence similarity, gene co-expression analysis employing genome-wide transcriptomics data across tissues or in response to environmental perturbation has become a valuable tool to predict gene function based on the GBA principle (Klie et al., 2010). The transfer of function annotations between two genes, exhibiting similar proﬁles, according to GBA is now a standard procedure for gene function prediction. Furthermore, gene co-expression networks have emerged as a powerful representative of the structure of similarity of transcriptomic proﬁles, and are readily employed for intra-species transfer of gene annotations following GBA (e.g., in the ﬁeld of plant science see, Obayashi et al., 2009; Mutwil et al., 2010; Mochida et al., 2011). Due to the previously described difference in the structure of GO and MapMan, we next evaluate the effect of these characteristics on the performance of gene function prediction by using a GBA-based network-driven approach. To this end, we employ a transcriptomic data-set of 273 publicly available Arabidopsis microarray experiments to construct a gene co-expression network (see Materials and Methods). We rely on the approach described in Mutwil et al. (2011) to obtain a co-expression network which is based on robust statistical parameter estimation combined with successive optimization of the biological relevance of the obtained network. In the co-expression network, the nodes correspond to Arabidopsis’ genes, and edges are established if the incident nodes (i.e., genes) are mutually in the top 30 most similar genes. The similarity is assessed by the Pearson correlation coefﬁcient, and this approach, termed highest reciprocal rank, has already been characterized to optimally capture functional annotation of co-expressed genes (Obayashi and Kinoshita, 2009). In the following, we rigorously extend this method to allow for a network-based prediction method of gene annotation by employing the method of majority voting (cf. Materials and Methods). In majority voting, the annotations of all adjacent nodes (i.e., immediate neighbors) of a given gene are ordered in a list, from the most to the least frequently appearing (Schiwikowski et al., 2000). The function of an unannotated gene is then predicted by the ﬁrst k functions in the list. Note that k is a user-speciﬁed parameter. Although the approach is very simple, it is exceptionally fast and can serve as an excellent reference for the amount of local information captured by the network due to the consideration of annotations of immediate neighbors. To generate and verify predictions of annotation with both ontologies, we conduct the following simulation: We ﬁrst select the genes which are annotated with concepts from each of the three ontologies, i.e., MapMan, GO-MF, and GO-BP, which resulted in 9,994 genes. Moreover, the annotation provided in all three ontologies for a set of randomly chosen genes is discarded. To June 2012 \| Volume 3 \| Article 115 \| 5 Klie and Nikoloski FIGURE 5 \| Distributions of concept depth in GO-MF (upper), GO-BP (middle), and MapMan (lower). The x -axis denotes the depth of a concept this end, the number of this artiﬁcially unannotated genes is set to be 4,000, corresponding to a fraction of ∼40% genes of unknown function. This scenario closely resembles the current state of Arabidopsis’ gene annotation. For these genes, prediction of gene annotation is obtained by using each one of the three ontologies. The predictions of the top k∈[1,20] most abundant concepts in the network vicinity are evaluated for their performance based on the original discarded annotation. For every k most abundant concepts from the unannotated genes, this procedure is repeated 1,000 times, such that in every iteration a different set of randomly unannotated genes is sampled. Note, that all three used ontologies were preprocessed so that for each gene all parent terms are included. Moreover, to avoid trivially correct predictions, such as the root terms of GO-MF and GO-BP, we do not consider the root terms as well the 20 less informative terms (based on the IC; see Materials and Methods). The predictions are summarized by precision and recall, two widely used performance measures in information retrieval and binary classiﬁcation Frontiers in Genetics \| Bioinformatics and Computational Biology Comparison of MapMan and GO while the left y -axis denotes the corresponding occurrence. For all three distributions, a normal distribution is ﬁtted (right y -axis). (Baeza-Yates and Ribeiro-Neto, 1999), as well as by their harmonic mean, the F -measure. On the other hand, we evaluate the biological relevance of the obtained predictions by investigating the normalized IC (with respect to the maximum) and the depth of the top k, k∈[1,20] predicted terms. Additionally, we also report the number of genes for which a prediction can be obtained following this procedure. Figure 8 summarizes the acquired prediction performance results for all three employed ontologies. One can observe that the use of MapMan exhibits an advantage in the performance of gene function prediction, as the combined F -measure is the highest over the whole range of top k, k∈[1,20] concepts (the exception is the case of k = 20, where the F-measure is zero, due to the lower number of terms in MapMan). This is mainly due to a higher average recall, i.e., a higher fraction of all the originally concepts, used in the annotation of a gene, that were successfully retrieved. Nevertheless, the average precision between GO and MapMan is comparable, indicating that the ratio of correctly June 2012 \| Volume 3 \| Article 115 \| 6 Klie and Nikoloski Comparison of MapMan and GO FIGURE 6 \| Histogram of the information content of all concepts used to annotate Arabidopsis’ genome by using the three ontologies MapMan, GO-MF, and GO-BP. predicted concepts to all predicted concepts is similar across all three ontologies. Correspondingly, the average IC and depth of concepts is generally higher in the case of MapMan, which implies a higher biological relevance or speciﬁcity of the predicted terms (Figure 8). However, both GO ontologies perform better with respect to the fraction of genes for which any prediction of gene annotation can be derived, i.e., 51% for MapMan vs. 64 and 73% for GO-MF and GO-BP, respectively. This suggests that the distribution of genome annotation is less clustered and more homogeneous. DISCUSSION Here, we provided the ﬁrst comparative analysis of two ontologies, GO, and MapMan, both widely used in plant biology studies. The ﬁrst part of the comparison comprises the structural characteristics of the ontologies, namely: the type of concepts and relationships between them as well as the design principles underlying GO and MapMan. Our ﬁndings were in support of the claim that higher level bins in MapMan correspond to terms of GO-BP, while lower level bins are more similar to terms of GO-MF. Regardless of these analogies, GO offers the possibility to also investigate gene products with respect to their spatial distributions, captured in the terms of the third GO ontology – cellular component (GO-CC). In contrast, MapMan does not www.frontiersin.org facilitate spatial analysis of genes and the downstream processes (e.g., metabolism). Nevertheless, although cellular processes and molecular functions are represented well in both GO and MapMan, temporal changes during plant development, fruit ripening, or progression of stress are in their nascent stages. Therefore, future developments in plant-speciﬁc ontologies should consider integrating the indicated spatial and temporal dimensions indispensable for accurate description of molecular processes in plants. In the second part of the study, we investigated the annotation corpus of Arabidopsis’ genes and carried out a detailed comparison of the two ontologies with respect to the information content of the respective concepts, i.e., bins in MapMan and terms in GO. It turned out that MapMan, GO-BP, and GO-MF exhibited similar relationships between information content and depth of concepts. In conjunction with the plethora of existing tools for computational analyses based on both ontologies, our results indicated that both ontologies may be equally suitable with respect to the biologically meaningful information that could potentially be extracted. Finally, we used the two ontologies as a principle source of information in the context of automated gene function prediction following the GBA principle on co-expression networks. The co-expression networks were created by using publicly June 2012 \| Volume 3 \| Article 115 \| 7 Klie and Nikoloski FIGURE 7 \| Visualization of information content (y -axis) at a given term depth (x -axis) for GO-MF (left), GO-BP (middle), and MapMan (right). A non-linear regression is used on the medians of the available transcriptomics data sets for Arabidopsis, and provided the medium for local propagation of concepts to unannotated genes in the vicinity of a given well-characterized gene. To this end, we used the simplest available alternative for automated function annotation given by the majority voting. Although our ﬁndings that MapMan outperformed GO with respect to function annotation depend on the algorithm for annotation transfer, we believe that they are robust as most of the available algorithms rely on propagation of local information only. While MapMan’s tree hierarchy at a ﬁrst glance appears to be ﬂatter, as assessed by term depth, and IC, in comparison to GO’s DAG structure, MapMan’s design tailored to Arabidopsis is most likely reﬂected in the improved performance in gene function prediction. In contrast to MapMan, GO represents a more generic ontology, reﬂected in its changing structure and gene annotation. Since no other plant model organism is currently equally well-annotated by GO and MapMan as it is the case for Arabidopsis, no general conclusions for plant species can be made. Nevertheless, what remains to be investigated is the effect of the distribution of annotated genes in the network. In other words, we expect that choice of the ontology for automated gene function annotation will ultimately depend on the dispersion of patches of annotated nodes (following the focused biological interest in genes of particular process/function). Frontiers in Genetics \| Bioinformatics and Computational Biology Comparison of MapMan and GO information content per concept depth following Michaelis-Menten kinetics (solid line). The constant V max /2 is shown by dotted lines (see main text for details). Last but not the least, the major implication of our study is that the choice of which ontology to be used computational analyses is problem-speciﬁc, as it highly depends on the interplay between the structural properties of the ontology, the size, and quality of the annotation corpus, using the ontology, as well as the employed multivariate data. Therefore, we believe that aside from the comparison of ontologies based on intra-ontology characteristics (e.g., distribution of information content), our study emphasizes the need for another criterion – namely, the biological question to be answered by using ontologies, for instance, comparison of plant developmental stages, or plant-speciﬁc structures and the here addressed gene annotation. This, of course, may open yet another ﬁeld of bioinformatics research related to the design of sound methods for ontology selection suitable for a particular problem at hand. In this respect, we believe that the suggested direction may result in development of (external and internal) measures for problem-speciﬁc comparison of ontologies and their performance – an issue which was already addressed in other research areas (e.g., data clustering, retrieval in audio and video databases). Taken altogether, the identiﬁed issues warrant caution in extending ontologies from model to other species and suggest that this may be most appropriately performed in a careful semi-automated manner. June 2012 \| Volume 3 \| Article 115 \| 8 Klie and Nikoloski FIGURE 8 \| Evaluation of prediction performance (y -axis) for gene function annotation using the three ontologies. Three classical performance indices from information retrieval were employed: F-measure (upper-left), precision (upper-middle), and recall (upper-right). In addition, the information content (lower-right), and the concept depth relative to the MATERIALS AND METHODS ARABIDOPSIS TRANSCRIPTOMICS DATA-SET AND RECONSTRUCTION OF A GENE CO-EXPRESSION NETWORK The employed transcriptomic data-set used to derive the gene co-expression network consist of 279 of publicly available microarray experiments (Affymetrix Ath1 gene-chip, 22,500 probe sets) obtained from the Gene Expression Omnibus2 (Edgar et al., 2002). Note, that this is the same transcriptomics compendium which is used in the PlaNet co-expression analysis platform (Mutwil et al., 2011). Initially, a total of over 6,000 microarray experiments were downloaded and the quality of each individual microarray experiment was ensured by an automated outlier detection and quality control. Here, the R Bioconductor package array Quality Metrics (Kauffmann et al., 2009) was employed to conduct (1) between-array comparisons based on distance between arrays and Principal Component Analysis, (2) 2 http://www.ncbi.nlm.nih.gov/geo/ www.frontiersin.org Comparison of MapMan and GO maximum depth within the respective ontology (lower-middle) of predicted concepts were evaluated. In all cases, the 20 most abundant concepts (x -axis) in an unknown gene’s neighborhood are evaluated. Finally, the fraction of unknown genes for which a prediction could be derived is given (bar plot, lower-right). inspection of array-wide probe intensity distributions by boxplots and density plots, (3) variance-mean dependence of each array, and (4) individual array quality assessment by MA plots. After this preprocessing, 1,707 microarrays were retained. Furthermore, this transcriptomics compendium was reduced by selecting a subset of experiments comprising 273 microarrays. This is performed to remove any bias arising through (potentially) un-informative or repetitive data while preserving the overall structure of the transcriptomics compendium (Mutwil et al., 2011). Brieﬂy, this selection strategy is based on the Subset Selection problem from linear algebra, whereby, for a given number l and a matrix A, one is to ﬁnd the subset of l columns from A which are most mutually independent. Here, columns of the matrix A denote individual microarray experiments (1,707 in total), rows correspond to genes, such that each matrix entry represents the corresponding gene expression levels. Application of the outlined selection procedure yielded 279 microarrays which were subsequently normalized using quantile normalization via the simple Affy R package. This data-set was used to reconstruct June 2012 \| Volume 3 \| Article 115 \| 9 Klie and Nikoloski the co-expression network and is available in the Table S1 in Supplementary Material. PREPROCESSING OF ONTOLOGIES – REMOVAL OF INCONSISTENCIES AND INTEGRATION OF PARENT CONCEPTS As sources of mapping genes to ontology terms in Arabidopsis, we employed the latest versions available for MapMan (Version 1.1 from January 2010)3 and GO (Version 2.5 from September 2010, available via the R package ath1121501.db4 ). Within these mappings, a total of 15,238 gene products are annotated with MapMan bins and 12,225 and 13,157 genes are annotated by GO-BP and GO-MF terms, respectively. However those raw mapping ﬁles contain inconsistencies: while the annotations for some genes contain only the most speciﬁc concepts, i.e., terminal or leaf concepts with no further child concepts, others are additionally annotated with parent concepts. As an example, consider the genes annotated with the MapMan bin “29.5.11.4.2” corresponding to “protein.degradation.ubiquitin.E3.RING” in Arabidopsis. This bin is a leaf or terminal concept, i.e., it has no children. One gene that is annotated with this concept is a member of the ARM repeat superfamily (locus ID at1g71020) and is additionally annotated with the parent bin “29.5.11” corresponding to “protein degradation ubiquitin.” However, other genes annotated with the bin 29.5.11.4.2, for instance EDA40 (at4g37890), are only annotated with the leaf bin “29.5.11.4.2” missing the mapping to any parent bins, e.g., 29.5.11.4 or 29.5.11. Likewise, similar examples hold for both GO domains, GO-MF, and GO-BP. In total, 25 of such inconsistencies can be identiﬁed for MapMan and 3,750 and 2,202 for GO-MF and GO-BP, respectively. The effect of an incomplete mapping which includes only partially – or even not at all – parent concepts is twofold: ﬁrst, the analysis by means of IC of a concept would lead to incorrect results since the IC of a concept is dependent on the number of genes associated with it. By deﬁnition of an ontology, a gene annotated with a low-level concept should automatically be annotated with all of the ancestral terms, too (Figures 1 and 9). Only considering the concept-gene association counts in a raw ontology will lead accidentally to erroneous results for the derived ICs; in this case leaf or terminal concepts might exhibit a higher IC than their parent terms (Klie et al., 2010). Second, for the purpose of gene function prediction in majority voting, common ancestor terms of the neighboring genes are of great importance. In the case that the annotation of all neighboring genes is a disjoint set of low-level concepts, no majority vote can be found (cf. Figure 9D). However, the gene’s neighbors can share common parent concepts that can help in deriving predictions for the gene in question. Although the derived annotation might not be as speciﬁc, the prediction of a high-level concept suggesting the putative involvement in processes or pathways is preferred to obtaining no prediction at all. To resolve the problem of incomplete mappings, we preprocessed all three ontologies so that for each gene, the complete list of parent terms is included. Note, that those parent terms can readily be identiﬁed by enumerating the respective DAG or tree structure deﬁned by is a or part of relations 3 http://mapman.gabipd.org/ 4 http://www.bioconductor.org Frontiers in Genetics \| Bioinformatics and Computational Biology Comparison of MapMan and GO (Figures 9A,B). We further deﬁne these modiﬁed mappings as “complete ontologies”. Finally, the preprocessing involved removal of control and unknown probe sets, which corresponds to probes associated with MapMan bins 0 and 35 (“control” and “unknown”/“not assigned”) and all their child bins. EVALUATION OF ONTOLOGY STRUCTURE AND INFORMATION CONTENT We employ two measures to characterize the structure and the characteristics of an ontology – the depth and the information content (IC) of concepts. Given a directed acyclic graph G = (V, E), which deﬁnes the relationships of concepts within an ontology, where V is a set of vertices, E is a set of edges, the depth of a term x is given by the distance d(x, r) between the two vertices x and r, where node r corresponds to the root concept of the ontology. Furthermore, the distance is deﬁned as the length of the shortest path from x to r (Bondy and Murty, 2008). Note that node r represents the root term which is explicitly deﬁned for GO-BP as and GO-MF and which can be implicitly deﬁned for MapMan by adding an artiﬁcial root node, i.e., bin r. The IC of an ontological concept c is deﬁned as IC(c) = log2 (\|Gc \|/\|G all \|), where Gc is the set of genes annotated with the concept c and G all is the set of genes annotated with any of the concepts in the ontology (Resnik, 1995). DETERMINING SIMILAR CONCEPTS ACROSS ONTOLOGIES To quantify the similarity of two concepts c 1 and c 2 , we use the Jaccard similarity coefﬁcient of the set of genes G 1 annotated with concept c 1 in MapMan and the set of genes G 1 annotated with concept c 2 in GO. The Jaccard similarity coefﬁcient for two sets G 1 and G 2 is deﬁned as sim(c 1, c 2 ) = J (G 1, G 2 ) = \|G 1 ∩G 2 \|/\|G 1 ∪G 2 \|. As 50% of all MapMan and GO concepts describe four or more genes, we consider only concepts of MapMan and GO that are annotated with at least four genes (i.e., \|G1 \| and \|G2 \| > 3) to avoid identifying similar concepts based on individual genes. In addition, to analyze the pair-wise similarity over all concepts, we create a network in which nodes correspond to concepts and edge are established between two nodes c1 and c2 if sim (c 1 , c 2 ) ≥ 0.6. Note, that despite its numerical value, this threshold is rather strict as it refers to only the highest 1% of all observed pairwise concept similarities, not only between MapMan and GO but also within the respective ontologies. Nodes corresponding MapMan bins that are not connected to a node denoting a GO term are discarded. Finally, the edges of the resulting network can be divided by the type of association between nodes they model: the similarity between a MapMan bin and GO-BP term, a MapMan bin and GO-MF term as well as a MapMan bin and both GO-MF and GO-BP terms. For each of those three derived types of associations, the average bin depth of MapMan bins is determined and the statistical signiﬁcance of the difference of means within the ﬁrst two groups (MapMan/GO-MF, MapMan/GO-BP) is derived via Wilcoxon-Rank-Sum test (Sokal and Rohlf, 2003). GENE FUNCTION PREDICTION USING NETWORK-BASED MAJORITY VOTING Majority voting is one of the simplest, yet fastest, network-based gene function prediction methods (Schiwikowski et al., 2000). June 2012 \| Volume 3 \| Article 115 \| 10 Klie and Nikoloski FIGURE 9 \| Preprocessing of ontologies and network-based gene function prediction by majority voting. (A) The original annotation of genes (“raw” annotation) and corresponding concepts (denoted by letters) is extended for each gene by including all parent concepts. The latter is referred to as “completed” annotation. Additional ﬁltering can be performed to remove concepts annotated by many genes (gray letters). (B) Parent terms can be readily obtained by traversal of the ontology structure (a node represents a concept, an arrow an is a or part of relationship among concepts; terminal or leaf concepts are denoted in black). (C) Gene Particularly, its reliance on the immediate neighborhood of a given node renders it applicable in estimating usefulness of local information on gene function prediction. Here, the network consists of nodes corresponding to the genes included in the aforementioned Arabidopsis transcriptomics compendium. The necessary steps to transform similarity of gene expression proﬁles to edges between genes in a ﬁnal co-expression network rely on the approach presented in Mutwil et al. (2011). In summary, this approach is comprised of ranking pair-wise gene expression proﬁles by the Pearson correlation coefﬁcient. Successively, the application of statistical tests is conducted to determine the optimal cut-off (range) for the reciprocal ranks which translate into establishing edges between the nodes in the network. Moreover, an optimality principle is employed to select a set of best-performing parameter values with respect to the GBA www.frontiersin.org Comparison of MapMan and GO function prediction of the unknown gene, denoted by I, by using the majority voting approach: the annotation of all immediate neighbors in a co-expression network (black ellipses) is considered. (D) Deriving a prediction for the gene I by ranking the annotation obtained through its neighbors. By using the raw annotation, unambiguous prediction cannot be derived (left column); the “completed” annotation aids in deriving meaningful predictions by considering concepts intermediate in the hierarchy (e.g., concept c, middle column); additional ﬁltering (right column) further improves the prediction (“optimized ontology”). principle. To this end, we conduct an iterative search on the allowable ranges for the reciprocal ranks that maximize the similarity of gene function in the neighborhood of a given gene/node. A highest reciprocal rank (HRR) cut-off between 10 and 30 produced biologically relevant networks (Mutwil et al., 2010). However, while >80% of the nodes were disconnected for HRR = 10, and consequently excluded from any further co-expression analysis, a HRR = 30 was chosen as the number of disconnected nodes decreased to 25%. Note, that by relying on ranks of derived from pair-wise correlations of gene expression proﬁles, no explicit threshold for the Pearson correlation coefﬁcient is needed. This is nicely illustrated by the range of Pearson correlation coefﬁcients of expression proﬁles of a pairs of genes with a HRR of 30 which varies from 0.32 to 0.9 depending on the individual gene. The advantage of using HHR rather than the simple pair-wise June 2012 \| Volume 3 \| Article 115 \| 11 Klie and Nikoloski correlation is that co-expression analysis by HRR uncovers more meaningful biological associations (Aoki et al., 2007). The obtained co-expression network is composed of 9,994 nodes, which correspond to those genes in Arabidopsis’ genome that are annotated with a set of concepts from all three ontologies, i.e., MapMan, GO-BP, and GO-MF. This network consists of 461 connected components of which 439 are singleton genes, i.e., nodes with no adjacent edges, and exhibits a density of 0.001. The largest component contains 9,506 nodes and the average degree of a node is 10.36. To simulate the effect on gene function prediction depending on the ontology used, the annotation provided in all three ontologies for a set of randomly chosen genes is discarded. To this end, the number of this artiﬁcially unannotated genes is set to be 4,000, a fraction corresponding to the ∼40% genes of unknown function in Arabidopsis. For each of the 4,000 genes, the annotations of all adjacent nodes are derived using the completed ontology and ordered in a list, separately for all three ontologies. Every concept present in the annotation of neighboring nodes is ranked from the most to the least frequently appearing within the neighborhood (Figure 9). The function of an unannotated gene is then predicted by examining the ﬁrst k functions in the list. Here, we consider the predictions of the top k∈[1,20] most abundant concepts in the FIGURE 10 \| Effect of the preprocessing of ontologies (cf. Figure 9) and the impact on network-based gene function prediction by majority voting for all three ontologies quantified by the F-measure (upper Frontiers in Genetics \| Bioinformatics and Computational Biology Comparison of MapMan and GO network vicinity and successively evaluate them by comparing the predicted terms to the original discarded annotation. This procedure is repeated 1,000 times, such that in every iteration a different set of randomly unannotated genes is sampled and evaluated for every k most abundant concepts. Furthermore, we removed those 20 concepts (corresponding to the choice of parameter k) with the lowest IC from all three complete ontologies. The aim of this ﬁltering step is to avoid deriving trivial annotation (e.g., the root concepts of the ontologies) or unspeciﬁc annotations (e.g., very broad, high-level biological concepts) as predictions. We note that although those high-level terms are technically correct in terms of prediction, their beneﬁt in characterizing a gene of unknown function is limited (cf. Figure 9D). An example of terms exhibiting a low IC are within the GO-BP sub-ontology “biological process” (GO:0008150), i.e. the root term or “transport” (GO:0006810). For GO-MF, examples of removed terms include “binding” (GO:0005488) and, again, the root node “molecular function” (GO:0003674). In contrast, more speciﬁc concepts of higher IC are unaffected by this ﬁltering step. These include, for instance, the children of the term “binding” which are “secretion” (GO:0046903) and “ion transport” (GO:0006811). These modiﬁed ontologies are termed “optimized ontologies” and further used for evaluation of the prediction panel) and the normalized depth of term/concept (lower panel) separately for raw, complete and optimized versions of the ontologies. June 2012 \| Volume 3 \| Article 115 \| 12 Klie and Nikoloski Comparison of MapMan and GO performance (Figure 8). Finally, the effect of this optimization step on gene function prediction is illustrated in Figure 10: A raw ontology only contains some of the ancestral concepts resulting in a lower prediction performance (F-measure; similar results hold for precision and recall; data not shown) and average term depth of predicted concepts (similar results hold for the average IC of predicted terms; data not shown). In contrast, the complete ontology includes all ancestral concepts deﬁned in the respective ontology, resulting in an increase of prediction performance; however, it is accompanied by a lower term depth of predicted concepts. The optimized ontology removes ambiguous terms, i.e., terms of high IC, and represents a compromise between good prediction performance and speciﬁcity of derived predictions. Interestingly, MapMan proﬁts the most from the proposed optimization strategy. EVALUATION OF GENE ANNOTATION PREDICTION PERFORMANCE The quality of the predicted ontological concepts for genes is evaluated by two complementary strategies. While the ﬁrst strategy comprises the use of classical quality measures from the ﬁeld of pattern recognition and information retrieval that assess the correctness of predicted terms, the second strategy seeks to quantify the quality of those derived predictions in terms of biological relevance. Again, the previously established concepts of term depth and IC are employed for this task. Note that for the purpose of comparative evaluation, both term depth and IC are normalized to the respective maximum value encountered within the particular ontology. REFERENCES Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., George, R. A., Lewis, S. E., Richards, S., Ashburner, M., Henderson, S. N., Sutton, G. G., Wortman, J. R., Yandell, M. 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Citation: Klie S and Nikoloski Z (2012) The choice between MapMan and Gene Ontology for automated gene function prediction in plant science. Front. Gene. 3:115. doi: 10.3389/fgene.2012.00115 This article was submitted to Frontiers in Bioinformatics and Computational Biology, a specialty of Frontiers in Genetics. Copyright © 2012 Klie and Nikoloski. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited. June 2012 \| Volume 3 \| Article 115 \| 14
W3003573439.txt	https://www.mdpi.com/2412-3811/5/2/14/pdf?version=1580478273	en		Characterizing the Performance of Ternary Concrete Mixtures Involving Slag and Metakaolin	Infrastructures	2,020	cc-by	9,265	infrastructures Article Characterizing the Performance of Ternary Concrete Mixtures Involving Slag and Metakaolin Matthew S. Sullivan, Mi G. Chorzepa * and Stephan A. Durham College of Engineering, The University of Georgia, Athens, GA 30602, USA; matthewseansullivan@hotmail.com (M.S.S.); sdurham@uga.edu (S.A.D.) * Correspondence: chorzepa@uga.edu Received: 17 December 2019; Accepted: 26 January 2020; Published: 31 January 2020 Abstract: Ternary blends of cementitious materials are investigated. A cement replacement level of 45% is used for all ternary mixtures consisting of 15% metakaolin and 30% slag replacements. Three metakaolin and two blast furnace slag, referred to as ‘slag’ for short, products commercially available are used to compare performance in ternary blends. A mixture with a 45% fly ash replacement is included to serve as a benchmark for performance. The control mixture contains 422 kg of cement per cubic meter of concrete, and a water-to-cementitious material ratio of 0.43 is used for all mixtures with varying dosages of superplasticizer to retain workability. Mixtures are tested for mechanical properties, durability, and volumetric stability. Mechanical properties include compression, split-cylinder tension, modulus of rupture, and dynamic Young’s modulus. Durability measures are comprised of rapid chloride-ion penetrability, sulfate resistance, and alkali–silica reactivity. Finally, the measure of dimensional stability is assessed by conducting drying shrinkage and coefficient of thermal expansion tests. Results indicate that ternary mixtures including metakaolin perform similarly to the control with respect to mechanical strength. It is concluded that ternary blends perform significantly better than both control and fly ash benchmark in tests measuring durability. Furthermore, shrinkage is reduced while the coefficients of thermal expansion are slightly higher than control and the benchmark. Keywords: metakaolin; blast furnace slag; ternary; strength; durability; concrete; supplementary cement; pozzolan; mineral admixtures; coefficient of thermal expansion 1. Introduction The use of supplementary cementitious materials (SCM) in ready-mixed concrete has seen rapid growth in North America since the 1970s [1]. Two types of chemical reactions contribute to the performance of hardened concrete mixtures incorporating SCMs. The first is the hydraulic reaction, which is the reaction of free lime (CaO) with water produces heat, calcium silicate hydrates (C–S–H), and calcium hydroxide, Ca(OH)2 or CH. In mixtures containing ordinary Portland cement (OPC), this reaction performed exclusively by the cement. However, some SCMs with significant CaO contents also take part in this reaction. Common SCMs take part in this reaction are Class C fly ash and blast furnace slag. The second reaction is the pozzolanic reaction, which is exhibited by SCMs with high contents of glassy silica or alumina in the presence of CH and water. In this reaction, amorphous (glassy) alumina or silica react with the CH created during hydration to produce additional C–S–H, calcium aluminate hydrates (C–A–H), or calcium aluminosilicate hydrates (C-A-S-H). In short, the additional pozzolanic and/or hydraulic reactions by SCMs tend to reduce porosity, consume non-structural CH (prone to leaching), and produce additional structural hydrates, all of which enhance concrete performance. Infrastructures 2020, 5, 14; doi:10.3390/infrastructures5020014 www.mdpi.com/journal/infrastructures Infrastructures 2020, 5, 14 2 of 17 Typically, SCMs are used as a cement-replacing material because of their ability to enhance performance, reduce costs, and reduce environmental impact. During the production of cement clinker, CaCO3 (or calcium carbonate) is converted into calcium oxide by means of calcination inside a rotary kiln [2]. The most environmentally detrimental byproduct, CO2 (carbon dioxide), is then emitted. Equation (1) presents the chemical reaction in a simplified form: CaCO3 + heat → CaO + CO2 (1) Cement production is the largest emitter of CO2 [3]. Furthermore, estimated production of the CO2 is 0.82 tons for each ton of cement produced [4] and amounts to 5% of the world’s anthropogenic carbon emissions [5]. Construction is still, however, a cost-plus business, and cement replacement continues to be prevalent because it reduces environmental and financial cost. Massive concrete elements typically utilize high levels of cement replacement by SCMs to combat high heats of hydration. For this application, Class F fly ash is most commonly used to replace approximately 50% of the cement, although slags are also effective when used at higher replacement levels [6]. Studies have found that the largest contributor to a reduction in the heat of hydration is the cement replacement level [7]. This effect is characterized by the reduction in heat associated with decreased hydraulic activity. By virtue of dilution, ternary blends of SCMs have also been proven to reduce the heat of hydration when replacing large amounts of cement [7–12]. In practice, however, binary replacements of cement by SCMs are typically used to combat this problem. 2. Background Generation of heat from hydration of cement and associated volume change in mass concrete operations have historically been combatted by replacing a large portion of cement by supplementary cementitious materials. Typically, the procedure is either Class F fly ash or ground granulated blast furnace slag replacing approximately between 30% and 50% of the cement. Although metakaolin has been present in concrete literature for decades, little work has been completed to explore its possible benefits inside ternary cementitious blends. Binary replacements by either fly ash or slag are effective in mitigating high heat of hydration, however, this approach is susceptible to material shortages. There is a tangible risk of the coal market being unable to sustain the current volume of fly ash usage in U.S concrete, particularly in the state of Georgia. Natural gas produces less emissions and is cheaper as a fuel source, and heightened regulations have increased the cost of collecting fly ash. Fly ash is an important SCM used in mitigating deleterious cracking due to the large thermal gradients in mass concrete, and the inability to obtain fly ash would have repercussions on mass concrete placements across the U.S. It is therefore important to evaluate alternative SCMs, as well as SCMs in ternary combinations. Recent research demonstrated the reduced heat liberation of ternary cementitious blends incorporating metakaolin and slags [8,9]. Studies have also shown increased performance of these mixtures in mechanical tests [13,14] and improved pore properties [15,16]. However, comprehensive studies on the behavior of ternary cementitious blends and their long-term performance (or durability) must be evaluated to determine the efficacy and limits of such usage in concrete mixtures. 3. Research Significance and Objectives Ternary mixtures including metakaolin have the potential of reducing the heat of hydration while maintaining mechanical strength, increasing the durability, and improving dimensional stability. While research has been conducted demonstrating the ability of ternary blends of metakaolin and slag to reduce the heat of hydration [9,10], comprehensive studies evaluating the mechanical properties, durability, and dimensional stability of these mixtures have not been conducted. Moreover, studies have not yet been conducted comparing the performance using various commercially-available metakaolin and blast furnace slag products. Blast furnace slag is for short referred to as ‘slag’ for the remainder of Infrastructures 2020, 5, 14 3 of 17 this study. This evidence would be crucial to the adoption of ternary blends of metakaolin and slag in ready-mix concrete operations, as product-dependent performance would present a risk in its usage. The research aims of this paper are two-fold: • • To determine if ternary blends of cementitious materials provide improved performance including durability and dimensional stability. To compare the performance of three commercially-available MKs in ternary cementitious blends. Therefore, the ultimate goals of this study are to explore the efficacy of a ternary metakaolin blends as an alternative to high volume fly ash, and secondarily to provide recommendations for its usage in concrete. 4. Materials and Experimental Plan Fly ash is a versatile SCM in that it is able to improve the fresh properties of concrete, increase durability, as well as reduce liberated heat in mass concreting [17]. It is for this reason that it is so widely used. The experimental study conducted in this paper will include a typical drilled shaft concrete design (mass pour) as a performance benchmark. As will be discussed, this mixture constitutes a 45% replacement of cement by class F fly ash. To control the dilution effect, ternary SCM combinations shall also replace a portion of cement equaling 45%. Literature indicates that the heat of hydration is minimized at a 45% replacement when replacements by metakaolin and slag are 15% and 30% of the cement content, respectively [9]. Therefore, all combinations of metakaolin and slag in this study shall be 15% and 30% of the cement content, respectively. Binary mixtures including 30% slag are also included to serve as additional benchmarks for performance. 4.1. Materials 4.1.1. Metakaolin Three commercially available metakaolin (MK) products are evaluated. The first (MK1), second (MK2), third product (MK3) is sourced from Sandersville, Georgia, Aiken, South Carolina, and Sandersville, Georgia, respectively. All MK products considered in this study are high-reactivity metakaolins (HRMs) that conform to ASTM C 618 [18]. The consensus within the literature is that a HRM should contain SiO2 + Al2 O3 + Fe2 O3 ≥ 90% by weight. As all MKs evaluated in this paper are high-reactivity with an average particle size of 1.3 µm (<1% residue on 45 µm sieve), they will simply be referred to as MKs. The reactivity of MK entirely depends on the kaolin’s purity, and the percentage of kaolinite that is dehydroxylated during calcination (amorphous alumina and silica). However, technical data sheets provided by the product vendors typically contain a breakdown of oxides by weight and material fineness. Thus, these two parameters are typically cited as predictors of performance. A combination of physical and chemical characteristics of the MK products selected in this study are found in Table 1. These properties are taken from products’ material specifications provided by manufacturers. Pictures of each product are presented in Figure 1, noting that the second product has a slightly brown coloration. Otherwise, the 3 products are difficult to visually tell apart. 4.1.2. Blast Furnace Slag Two, grade 120, commercially available slag (SL) products were used in this study. They are denoted as ‘SLa’ and ‘SLb’. As with metakaolin products, the chemical composition and physical characteristics are important in understanding slag behavior once inside the concrete and are presented in Table 1. Infrastructures 2020, 5, 14 4 of 17 Table 1. Chemical and physical properties of materials. Property Composition Cement MK1 SiO_2 (%) 19.70 Al_2O_3 (%) 4.70 Fe_2O_3 (%) 3.00 Infrastructures 2020, 5, x FOR PEER REVIEW CaO (%) 63.30 Chemical MgO (%) 3.10 Na_2O 0–<0.12.70 LOI(%) (%) TiO_2 (%) 0–<0.1 Specific Surface (BET), 3.20 SO_3 (%) 387 /kg SpecificmGravity LOI C (%) 3S Specific Surface (BET), m2 /kg 2 Physical Bogue Bogue C3C S 2S C2 S C3C A3A C4 AF C4AF 3.16 2.70 387 54 15 7 9 54 15 7 MK2 MK3 SLa SLb FA 50.75 54–56 51.66 33.33 34.07 45.91 40–42 43.99 13.50 12.73 0.45 <1.4 0.47 0.68 0.47 0.06 <0.1 0.01 41.28 40.41 0.00 <0.1 0.03 5.53 6.56 0.23 0.42<0.05 <1.0 1.89 0.50 0.21 2.80 0.172.87 1.87 <3 1.89 1.89 0.52 0.08 14,200 <0.0520,000 - 11,0002.30 472 * 531 50.77 26.65 3.76 4 of 19 11.30 2.15 1.17 2.53 * 1.20 430 2.60 0.42 14,200 2.50 2.53430 - - 2.60 <1.0 20,000 - - 2.50 0.50 11,000 - - * 2.80 472 - - - 1.05 2.87 531 - - - - 9 Note: * Material data not provided; - Material data not applicable. Note: * Material data not provided; - Material data not applicable. Figure 1. 1. Sample Sample of of metakaolin metakaolin products products (a) (a) MK1; MK1; (b) (b) MK2; MK2; (c) (c) MK3. MK3. Figure 4.1.3. Fly Ash and Cement 4.1.2. Blast Furnace Slag The fly ash (FA) is an ASTM C618 [18] Class F FA and is a raw material (<34% retained on the Two, grade 120, commercially available slag (SL) products were used in this study. They are 0.044 mm sieve). Pertinent physical and chemical properties for the FA is also included in Table 1. denoted as ‘SLa’ and ‘SLb’. As with metakaolin products, the chemical composition and physical Finally, a Type I/II Portland cement conforming to ASTM C150 [19] was used for all concrete and characteristics are important in understanding slag behavior once inside the concrete and are mortar mixtures. The cement is local to Atlanta, Georgia. Table 1 lists the physical and chemical presented in Table 1. characteristics, as well as the Bogue phase composition. 4.1.3. Ash andand Cement 4.1.4. Fly Aggregates Chemical Admixtures The fly ash conforming (FA) is an ASTM C618 F FAin and is a raw and material (<34% retained the Aggregates to ASTM C33[18] [20]Class were used all concrete mortar mixtures. The on coarse 0.044 mm sieve). Pertinent physical and chemical properties for the FA is also included in Table 1. and fine aggregates are sourced from Watkinsville, Georgia and Athens, Georgia, respectively. Neither Finally, a Type I/II Portland cement The conforming to ASTM is C150 [19] granite was used for all concrete aggregate is known to be reactive. coarse aggregate graded stone, and the sizeand of mortar mixtures. The cement is local to Atlanta, Georgia. Table 1 lists the physical and aggregate used is the standard size #57 (NMAS 1” or 2.54 mm). The fine aggregate materialchemical contains characteristics, as well the Bogue phase 100% of particles that as passes the 9.5 mm composition. (3/8 inch) sieve and less than 3% that passes the 75 µm (No. 200) sieve. Water used for mixing was potable tap water. Finally, a PCE-based (polycaboxylate 4.1.4. Aggregates and Chemical Admixtures ether) superplasticizer was used for the mixtures (33% solids). Aggregates conforming to ASTM C33 [20] were used in all concrete and mortar mixtures. The 4.2. Mixture Design coarse and fine aggregates are sourced from Watkinsville, Georgia and Athens, Georgia, respectively. Neither aggregate is known to be reactive. is graded granite stone, and the size Concrete mixture proportions are basedThe on acoarse drilledaggregate shaft design that incorporates a high volume of of usedofishydration the standard sizeThe #57mixture (NMASdesign 1” oris2.54 mm). The 2. fine material FA aggregate to reduce heat and cost. found in Table Theaggregate total cementitious 3 . The contains 100% particles passes the 9.5 mm (3/8 inch) sievematerial and less(w/cm) than 3% thatispasses thethe 75 content for eachofmixture is that 422 kg/m water-to-cementitious ratio 0.43, and 3 µm (No. 200) sieve. Water mixing was potableincorporate tap water. Finally, a PCE-based coarse aggregate fraction is 753used kg/mfor . All concrete mixtures a target air content equal (polycaboxylate ether) superplasticizer was used for the mixtures (33% solids). to 4%. SCMs replace a percentage of cement by weight. The weight of sand varied by mixture and 4.2. Mixture Design Concrete mixture proportions are based on a drilled shaft design that incorporates a high volume of FA to reduce heat of hydration and cost. The mixture design is found in Table 2. The total cementitious content for each mixture is 422 kg/m3. The water-to-cementitious material (w/cm) ratio Infrastructures 2020, 5, 14 5 of 17 was determined according to the absolute volume method as in ACI 211. Saturated surface-dry (SSD) mixture proportions are listed in Table 2. Table 2. Saturated surface-dry (SSD) proportions per m3 of mixture. Mixture Cement (kg) Meta-Kaolin (kg) Slag (kg) Fly Ash (kg) Coarse Aggregate (kg) Fine Aggregate (kg) Water (kg) Air (%) Super-Plasticizer (mL/kg.cm) Control SLa-30 SLb-30 FA-45 MK1-15_SLa-30 MK1-15_SLb-30 MK2-15_SLa-30 MK2-15_SLb-30 MK3-15_SLa-30 MK3-15_SLb-30 422 295 295 232 232 232 232 232 232 232 0 0 0 0 63 63 63 63 63 63 0 126 126 0 126 126 126 126 126 126 0 0 0 190 0 0 0 0 0 0 985 985 985 985 985 985 985 985 985 985 723 713 713 663 702 702 702 702 702 702 182 182 182 182 182 182 182 182 182 182 4 4 4 4 4 4 4 4 4 4 2.2 2.2 2.7 0.5 4.8 4.8 4.6 4.8 4.6 4.6 Mortar specimens were batched and tested according to ASCM C1012 [21] and C1567 [22] for sulfate reactivity and alkali–silica reactivity, respectively. Mixture proportions differed for these tests as per the testing standards. For the sulfate mixtures, cementitious material to sand ratios were 1:2.75 and w/cm ratios were 0.485. Cement replacements by SCMs were by weight as in the concrete mixtures. Finally, a superplasticizer was included to control for significant workability difference between mixtures. This deviation from ASTM C1012 [21] was felt necessary by the authors, as the loss of concrete performance with increasing w/cm is exaggerated with metakaolin inclusion [23]. ASR specimens were created according to specific batch weights. A 990 g quantity of sand was mixed with 440 g of total cementitious material. W/cm ratios were 0.43. Workability was maintained amongst mixtures by inclusion of a superplasticizer. 4.3. Batching and Molding Concrete mixtures were machine mixed using a portable revolving drum mixer with a 0.35 m3 capacity. Saturated surface dry (SSD) mixture proportions were altered according to the volumetric method to accommodate the moisture condition of the aggregates upon mixing, and then weighed using a scale with a 136 kg capacity and 0.05 kg accuracy. Materials were added and mixed per ASTM C192 [24]. The superplasticizer was added to the mixing water and thoroughly agitated before the water was added to the mixer. Contents of the mixer were then discharged into a wheelbarrow so that fresh concrete tests could be performed, and molds were filled. Material from the same batch filled the specimen molds for all hardened concrete tests excluding sulfate and ASR. Mortar mixtures were mixed using a portable table-top mixer with a nominal capacity of 4.8 L, and according to ASTM C305 [25] mixing procedure. The superplasticizer was added separately after adding water as the paddle rotated. The plastic mixture was then discharged into molds. 4.4. Concrete Mixture Properties Testing and Procedure 4.4.1. Fresh Properties After batching, the temperature, air content, slump, and unit weight were measured. The tests for unit weight and air were per ASTM C138 [26] and C231 [27], respectively, while slump was taken per C143. Temperature was measured with a thermometer per ASTM C 1064 [28]. 4.4.2. Mechanical Properties Specimens were made according to ASTM C192 [24], and were subjected to curing in a lime bath before testing. Compression cylinders measuring 100 mm × 200 mm were made for 1, 7, and 28-day strengths. Mixture strengths were taken as the average of three specimen strengths at each testing Infrastructures 2020, 5, 14 6 of 17 day. The universal testing machine used to break the cylinders had a capacity of 3.5 MN, which used neoprene caps for restraints. Cylinders were subject to a loading of 2000 N/s, per ASTM C39 [29]. Tensile strength for concrete was measured two ways: split-cylinder, and MOR. The split cylinder tests were performed on cylinders measuring 100 mm × 200 mm, while the MOR prisms were 150 mm × 150 mm × 550 mm. The testing protocol for each were per ASTM C 496 [30] and C78 [31], respectively. The same universal testing machine was used for the tensile tests as the compressive tests. Specimens were loaded at a constant rate of 130 N/s. The mixture tensile strengths were the average of three individual specimen strengths at the age of 28 days. The dynamic modulus of elasticity (Ed ) test was performed on cylinders measuring 100 mm × 200 mm at 28 days of age. Testing equipment measures concrete resonant frequency in the longitudinal direction, which was achieved by the forced resonance method outlined in ASTM C215 [32]. 4.4.3. Durability Tests including Permeability, Sulfate Attack, and ASR Tests of durability performed on concretes follow the same laboratory mixing and curing protocol as the specimens in strength testing. Rapid chloride-ion penetrability tests at 28 days of age were used as an indirect measure of the permeability of each SCM combination. Top sections of concrete cylinders (100 mm × 50 mm) were cut for testing, as this is the most permeable section. RCPT testing lasted for six hours per the ASTM C1202 [21]. The average charge passed by two specimens was taken as the permeability of the mixture. Protocol outlined in ASTM C1012 [21] was used to test for sulfate resistance of mixtures. Mortar cubes measuring a side length of 50 mm were batched alongside mortar prisms measuring 25 mm × 25 mm × 280 mm. Both were stored for 24 h at a temperature of 95 ◦ F. Periodically, the cubes would be tested for compressive strength until the average compressive strength of any two specimens exceeded 20 MPa. The mortar prisms were immersed in plastic containers of aqueous solutions of Na2 SO4 . Each mortar bar was subjected to exposure to 0.75 L of solution, at a concentration of 50 g/L Na2 SO4 . Expansions of the bars were measured at 7, 14, 21, 28, 56, 91, 105, 126, and 168 days in solution. The expansion of each mixture was taken as the average of six specimens. At this concentration, the expansion limit considered to effectively mitigate sulfate attack is 0.1%. For each ASR mixture, three mortar prisms (same dimensions as sulfate prisms) were batched and burlap cured at 23 ◦ C for 24 h. At the end of the 24-h curing period, the mortar bars were released from the molds and immersed in tap water elevated to 80 ◦ C. This temperature was maintained for 24 h. After this period, mortar bars were immersed in an aqueous solution of 40 g/L maintained at 80 ◦ C. Expansion readings were taken at 1, 5, 9, and 14 days after immersion in solution. As with the sulfate test, the maximum expansion that can be interpreted as mitigating the expansion is 0.1%. 4.4.4. Dimensional Stability Tests including CTE and Drying Shrinkage The coefficient of thermal expansion (CTE) was measured on 100 mm × 175 mm concrete cylinders. The testing followed the protocol outlined in AASHTO T336, which subjected the concrete to three heating and cooling cycles between 10 ◦ C and 50 ◦ C. An LVDT with a sensitivity of 0.001 mm detected changes in length as a function of temperature. The average strain per unit temperature change of three specimens was taken as the CTE of any given SCM combination. Drying shrinkage over a period of nine months was measured for each mixture at an ambient temperature of 22 ◦ C and a relative humidity of 50%, after an initial 28-day curing period in a lime-saturated bath. The protocol, which is in accordance with ASTM C157 [33], utilized concrete prisms of dimension 100 mm × 100 mm × 280 mm to measure the average shrinkage of three specimens per mixture. Each prism was suspended from the shelf, and rested on two wooden blocks at either end. Measurements of length change were taken at 32, 25, 42, 56, 84, 140, and 252 days after first batching. Infrastructures 2020, 5, 14 7 of 17 Infrastructures 2020, 5, x FOR PEER REVIEW 7 of 19 5.mixture. Results Each prism was suspended from the shelf, and rested on two wooden blocks at either end. Measurements of length change were taken at 32, 25, 42, 56, 84, 140, and 252 days after first batching. 5.1. Fresh Properties 5. Results Table 3 shows the fresh concrete properties for all concrete mixtures. Slumps ranged from 25 mm (1 inch) to 250 mm (10 inches), and air content typically ranged from 2.6% to 5.5%. Placement 5.1. Fresh Properties temperatures ranged between 24 ◦ C (75 ◦ F) and 30 ◦ C (86 ◦ F). Table 3 shows the fresh concrete properties for all concrete mixtures. Slumps ranged from 25 Table 3. Fresh concrete mixture properties. mm (1 inch) to 250 mm (10 inches), and air content typically ranged from 2.6% to 5.5%. Placement temperatures ranged between 24 °C (75 °F) and 30 °C (86 °F). Plasticizer Mixture Control SLa-30 Mixture SLb-30 FA-45 Control MK1-15_SLa-30 MK2-15_SLa-30 SLa-30 MK3-15_SLa-30 SLb-30 MK1-15_SLb-30 MK2-15_SLb-30 FA-45 MK3-15_SLb-30 MK1-15_SLa-30 Slump (cm) 7.6 Slump 2.5 2.5 (cm) 15.2 7.6 25.4 4.4 2.5 22.9 2.5 22.9 9.7 15.2 5.1 25.4 MK2-15_SLa-30 4.4 5.2. Mechanical Properties MK3-15_SLa-30 22.9 5.2.1. Compressive Strength MK1-15_SLb-30 22.9 Air Content Unit Weight Dosage (%) (kg/m3 ) (mL/kg.cm) Table 3. Fresh concrete mixture properties. 4.1% 2346 2.15 Air Content Plasticizer Dosage 4.3% Unit Weight 2192 4.77 4.56 (%) 3.0% (kg/m3)2353 (mL/kg.cm) 0.0% 2340 0.50 4.1%4.5% 2346 2253 2.15 4.77 3.9% 2266 2.19 4.3% 2192 4.77 4.5% 2186 2.67 3.0%3.5% 2353 2317 4.56 0.54 3.8% 2301 4.76 0.0% 2340 0.50 5.0% 2321 4.55 Temperature (◦ C) 23 Temperature 20 28(°C) 30 29 23 23 20 24 29 28 29 30 27 4.5% 2253 4.77 29 3.9% 2266 2.19 23 4.5% 2186 2.67 24 3.5% 2317 0.54 29 Strength evolutions in Figure 2. MK2-15_SLb-30 9.7for all mixtures 3.8% are found 2301 4.76 29 Of the two binary mixtures including slag or fly ash, only one mixture achieved a 28-day strength MK3-15_SLb-30 5.1 5.0% 2321 4.55 27 higher than control. The results from slag binary mixtures are presented to see the effect of replacing additional 15% of MK with cement. SLb-30 exhibited a 28-day compressive strength of 64 MPa 5.2. Mechanical Properties (9222 psi), an increase of 18%. Mixtures SLa-30 and FA-45 were 2% and 51% weaker in compression than the control, respectively. 5.2.1. Compressive Strength The rates of strength gain were similar between the two binary slag mixtures, the exception of the results being that SLb-30 was stronger than SLa-30 by slightly over Strength evolutions for all mixtures are found in Figure 2. 9 MPa (1300 psi) at one day of age. This was nearly the same difference in strength at 28-days of age. (a) Figure 2. Cont. Infrastructures 2020, 5, x 14FOR PEER REVIEW of 19 17 88of (b) Figure 2. strength evolution for for (a) non-metakaolin binary SCM,SCM, and (b) SCM 2. Compressive Compressive strength evolution (a) non-metakaolin binary andternary (b) ternary SCM mixtures. mixtures. Of the ternary mixtures, onlyincluding one mixture higher compressive strengthathan the two binary mixtures slaghad or afly ash, 28-day only one mixture achieved 28-day control. higher MK1-15_SLa-30 finished with afrom compressive strength of are 59 presented MPa (8570topsi), 10%effect higher strength than control. The results slag binary mixtures see the of than control. MK3-15_SLa-30 finished with a compressive strength very near control, with 54 MPa replacing additional 15% of MK with cement. SLb-30 exhibited a 28-day compressive strength of 64 (7770 (9222 psi). psi), One-day strengthsofwere or less thanand control forwere all ternary concrete mixtures MPa an increase 18%.similar Mixtures SLa-30 FA-45 2% and 51% weaker in including SLa. Their strengthsThe ranged 5 MPa (715 7MPa (1010 psi).the Apart compression than thecompressive control, respectively. ratesfrom of strength gain psi) weretosimilar between two from MK1-15_SLa-30, the 7-day strength forresults concrete mixtures including were lower than both binary slag mixtures, the exception of the being that SLb-30 wasSLa stronger than SLa-30 by control and including SLb. However, an age of nearly 7 days,the all SLa concrete mixtures slightly overthose 9 MPa (1300 psi) at one day of after age. This was sameternary difference in strength at gained compressive strength at a higher rate than did SLb ternary mixtures and the control. 28-days of age. By contrast, ternary mixtures SLb did suffer lower strengths,strength ranging than between Of the ternary mixtures, onlyincluding one mixture hadnot a higher 28-day1-day compressive the 12 MPa and 14.5 MPa (1730 psi and 2110 psi), thoughstrength they were still lower than control. Moreover, control. MK1-15_SLa-30 finished with a compressive of 59 MPa (8570 psi), 10% higher than ternary mixtures including SLb exhibited the highest rate of strength gaincontrol, from 1–7with days54but saw less control. MK3-15_SLa-30 finished with a compressive strength very near MPa (7770 dramatic strength gain after 7 days of age. Overall (excluding themixtures 28-day strength for psi). One-day strengths were similar or less than control for MK1-15_SLa-30), all ternary concrete including ternary mixtures rangedstrengths from 48 MPa (6970 psi)5 to 54 MPa (7769 SLa. Their compressive ranged from MPa (715 psi) topsi). 7MPa (1010 psi). Apart from MK115_SLa-30, the 7-day strength for concrete mixtures including SLa were lower than both control and 5.2.2. Tensile Strength those including SLb. However, after an age of 7 days, all SLa ternary concrete mixtures gained compressive strength at aresults higherofrate than didand SLbflyternary mixtures and the The tensile strength binary slag ash concrete mixtures, ascontrol. well as ternary concrete By contrast, mixtures including SLb did not suffer lower 1-day strengths, ranging mixtures are foundternary in Figure 3. between 12 MPa 14.5 MPa psi and 2110 psi), though they were still SLa-30 lower than control.a Binary slag and mixtures were(1730 stronger in tension than control. Mixture produced Moreover, ternary mixtures including SLblower exhibited highest rate of strength from 1–7 days splitting-tensile strength that was 15% thanthe control, while SLb-30 wasgain 17% higher than but saw less dramatic strength after 7mixtures days of were age. Overall (excluding MK1-15_SLa-30), the 28control. MOR values for thesesgain concrete both higher than control, ranging between day strength for ternary from 48The MPa psi) to 54 MPa (7769 psi). reduction in 5.1 and 6.6 MPa (740 andmixtures 958 psi),ranged respectively. fly(6970 ash mixture yielded a strength splitting-tension and MOR of 24% and 23%, respectively. Ternary SCM mixtures typically displayed 5.2.2. lowerTensile tensile Strength strength than control, however, MK1-15_SLa-30 performed similarly to control for both split-tension and MOR. Furthermore, theslag exception of MK2-15_SLb-30 split tension results, ternary The tensile strength results of with binary and fly ash concrete mixtures, as well as ternary mixtures incorporating MK1 and MK3 appear to have higher tensile strengths than do ternary mixtures concrete mixtures are found in Figure 3. incorporating MK2. Finally, the choice of slag product does not appear to negatively affect the tensile strength of concrete in ternary mixtures. Infrastructures 2020, 5, 14 9 of 17 Infrastructures 2020, 5, x FOR PEER REVIEW 9 of 19 Figure 3. Tensile strength of non-metakaolin binary and ternary SCM mixtures. Figure 3. Tensile strength of non-metakaolin binary and ternary SCM mixtures. 5.2.3. Dynamic Modulus of Elasticity Binary slag mixtures were stronger in tension than control. Mixture SLa-30 produced a splitting- tensile mixtures strength that was 15% of lower thanSCM control, while SLb-30 was 17% higher than control. MOR Concrete consisting binary mixtures (non-metakaolin) and ternary SCM mixtures values for theses concrete mixtures were both higher than control, ranging between 5.1 and 6.6 MPa have their modulus values given in Figure 4. Binary slag concrete mixtures displayed higher dynamic (740 and 958 psi), respectively. The fly ash mixture yielded a strength reduction in splitting-tension Young’s and moduli (E ) than the control, while all other mixtures saw a reduction from control. Increases MOR ofd 24% and 23%, respectively. Ternary SCM mixtures typically displayed lower tensile were onstrength the order 1 GPa and 0.4 GPa (150 ksi and 60 similarly ksi) for toSLa-30 SLb-30, respectively. thanof control, however, MK1-15_SLa-30 performed control and for both split-tension The greatest reduction in thewith Young’s modulus was exhibited bytension the flyresults, ash mixture, a decrease of and MOR. Furthermore, the exception of MK2-15_SLb-30 split ternary mixtures incorporating MK1 were and MK3 appear totrends have higher strengths ternary mixtures 28% from control. There no obvious amongtensile metakaolin orthan slagdo products for the ternary incorporating MK2. Finally, the choice of slag product does not appear to negatively affect the tensile concrete mixtures, and reductions in Ed ranged from 12%-22% with an average reduction of 5.6 MPa strength of 2020, concrete in PEER ternary mixtures. Infrastructures 5, x FOR REVIEW 10 of 19 (811 ksi). 5.2.3. Dynamic Modulus of Elasticity Concrete mixtures consisting of binary SCM mixtures (non-metakaolin) and ternary SCM mixtures have their modulus values given in Figure 4. Binary slag concrete mixtures displayed higher dynamic Young’s moduli (Ed) than the control, while all other mixtures saw a reduction from control. Increases were on the order of 1 GPa and 0.4 GPa (150 ksi and 60 ksi) for SLa-30 and SLb-30, respectively. The greatest reduction in the Young’s modulus was exhibited by the fly ash mixture, a decrease of 28% from control. There were no obvious trends among metakaolin or slag products for the ternary concrete mixtures, and reductions in Ed ranged from 12%-22% with an average reduction of 5.6 MPa (811 ksi). Figure 4. Dynamic Young’s modulus of non-metakaolin, binary and ternary SCM mixtures. Figure 4. Dynamic Young’s modulus of non-metakaolin, binary and ternary SCM mixtures. 5.3. Durability 5.3.1. RCPT Figure 5 shows that only one of three binary SCM concrete mixtures were able to reduce chloride-ion permeability below control. SLa-30 performed similarly to control, while FA-45 nearly Infrastructures 2020, 5, 14 10 of 17 5.3. Durability 5.3.1. RCPT Figure 5 shows that only one of three binary SCM concrete mixtures were able to reduce chloride-ion permeability below control. SLa-30 performed similarly to control, while FA-45 nearly reached the ‘high’ penetrability classification. SLb-30, however, was effective in reducing the total charge passed to 1440 Coulombs (C), earning a ‘low’ penetrability classification. All ternary SCM concrete mixtures fell below 1000 total coulombs passed. Four mixtures were under 500 C. Penetrability classes and results Infrastructures 2020, 5, x FOR PEER REVIEW 11 of 19 for non-metakaolin binary SCM mixtures and ternary SCM concrete mixtures are shown in Figure 5. Figure 5. Permeability of non-metakaolin binary and ternary SCM mixtures by RCPT. Figure 5. Permeability of non-metakaolin binary and ternary SCM mixtures by RCPT. 5.3.2. Sulfate Resistance 5.3.2. Sulfate Resistance Sulfate test results are presented in Figure 6. Fly ash was the only SCM other than metakaolin that wasSulfate able totest sufficiently mitigate mortar expansion thewas entire testing In fact, results are presented in Figure 6. Flyfor ash the6-month only SCM otherperiod. than metakaolin expansion by the mitigate binary slag mortars was more accelerated than the control mixture. that wasexperienced able to sufficiently mortar expansion for the entire 6-month testing period. In fact, This was especially true forby thethe mortar incorporating SLa. This is attributed to SLa both higher expansion experienced binary slag mortars was more accelerated thanhaving the control mixture. Al2This O3 content and thetrue lower Although the expansion by the binary mixtures are was especially forfineness. the mortar incorporating SLa. Thisseen is attributed to SLaslag having both higher excessive, it has been recommended that a slag be used at 50% or higher replacement level to mitigate Al2O3 content and the lower fineness. Although the expansion seen by the binary slag mixtures are sulfate attackit[34]. In fact, for high Al2 O replacement levels level of above 60% excessive, has been recommended that a slag be(14% usedoratgreater), 50% or higher replacement to mitigate 3 contents aresulfate recommended for this usefor [35,36]. ternary mixtures able to replacement mitigate the sulfate expansion, attack [34]. In fact, high All Al2O 3 contents (14% were or greater), levels of above 60% andare typically resulted in than expansion over 6 mixtures months. were able to mitigate the sulfate recommended forless this use0.06% [35,36]. All ternary expansion, and typically resulted in less than 0.06% expansion over 6 months. 5.3.3. ASR Resistance All ternary mortars resulted in expansions that were less than 0.03%. Results are presented in Figure 7. As with the sulfate tests, binary slag mortars at a replacement level of 30% were not sufficient Infrastructures 2020, 5, 14 11 of 17 to limit the expansion to below acceptable limits (expansions of 0.11% and 0.12%). The binary fly ash Infrastructures 2020, 5, x FOR PEER REVIEW 12 of 19 mortar exhibited similar expansion to the ternary mixtures. Figure 6. 2020, Expansion ofof non-metakaolin binary and ternary mortar mixtures in sulfate sodium Figure 6. Expansion non-metakaolin binary and ternary SCMSCM mortar mixtures in sodium Infrastructures 5, x FOR PEER REVIEW 13 of 19 sulfate solution. solution. 5.3.3. ASR Resistance All ternary mortars resulted in expansions that were less than 0.03%. Results are presented in Figure 7. As with the sulfate tests, binary slag mortars at a replacement level of 30% were not sufficient to limit the expansion to below acceptable limits (expansions of 0.11% and 0.12%). The binary fly ash mortar exhibited similar expansion to the ternary mixtures. Figure 7. Expansion of non-metakaolin binary and ternary SCM mortar mixtures in sodium Figure 7. Expansion of non-metakaolin binary and ternary SCM mortar mixtures in sodium hydroxide solution. hydroxide solution. 5.4. Dimensional Stability 5.4.1. Drying Shrinkage Figure 8 indicates that the ultimate drying shrinkage of ternary concrete mixtures containing MK and SL products were predominantly lower than the control, with an exception of MK1-15_SLa30. The 224-day drying shrinkage of the control mixture was 0.048%. One mixture, FA-45 was slightly Infrastructures 2020, 5, 14 12 of 17 5.4. Dimensional Stability 5.4.1. Drying Shrinkage Figure 8 indicates that the ultimate drying shrinkage of ternary concrete mixtures containing MK and SL products were predominantly lower than the control, with an exception of MK1-15_SLa-30. The 224-day drying shrinkage of the control mixture was 0.048%. One mixture, FA-45 was slightly larger at 0.052%. The overall drying shrinkage of ternary mixtures decreased with the binary mixture, SLb, showing the lowest shrinkage until 112 days. For ternary mixtures including MK1, these values decreased to 0.034% and 0.042% for MK1-15_SLa-30 and MK1-15_SLb-30 mixtures, respectively. For ternary mixtures including MK2, shrinkage values decreased to 0.037% and 0.034% for MK2-15_SLa-30 and MK2-15_SLb-30, respectively. Similarly, ternary mixtures containing MK3, concrete mixtures decreased to 0.037% (in MK3-15_SLa-30) or 0.034% (in MK3-15_SLb-30), which is Infrastructures 2020, 5, x FOR PEER REVIEW 14 of 19 about 30% lower than the control. Figure 8. Drying shrinkage of alternate binary and ternary SCM mixtures. Figure 8. Drying shrinkage of alternate binary and ternary SCM mixtures. 5.4.2. Coefficient of Thermal Expansion (CTE) 5.4.2. Coefficient of Thermal Expansion (CTE) CTE results are presented in Figure 9. The binary fly ash concrete exhibited a 2% lower CTE than control. SLa-30are and SLb-30 varied widely frombinary one another 3% and 7% higher than control, CTE results presented in Figure 9. The fly ashatconcrete exhibited a 2%the lower CTE than respectively. wasvaried the highest CTEfrom seen outside of the binary concrete mixtures. control. SLa-30The andlatter SLb-30 widely one another at 3% metakaolin and 7% higher than the control, ◦C MK1-15_SLa-30 saw the CTE inCTE the seen ternary groupofwith CTE of 9.73 × 10−6concrete mm/mm/ respectively. The latter waslowest the highest outside the abinary metakaolin mixtures. ◦ an increase of 1% from control). MK2-15_SLb-30 saw the highest CTE in the (5.40 × 10−6 in/in/ MK1-15_SLa-30 sawF,the lowest CTE in the ternary group with a CTE of 9.73 × 10−6 mm/mm/°C (5.40 ternary group with a CTE of 10.25 × 10−6 mm/mm/◦ C (5.70 × 10−6 in/in/◦ F, an increase of 7%). As a × 10−6 in/in/°F, an increase of 1% from control). MK2-15_SLb-30 saw the highest CTE in the ternary group the ternary SCM mixtures averaged a CTE of 9.93 × 10−6 mm/mm/◦ C (5.53 × 10−6 in/in/◦ F, −6 −6 group with a CTE of 10.25 × 10 mm/mm/°C (5.70 × 10 in/in/°F, an increase of 7%). As a group the an increase of 3%). ternary SCM mixtures averaged a CTE of 9.93 × 10−6 mm/mm/°C (5.53 × 10−6 in/in/°F, an increase of 3%). control. SLa-30 and SLb-30 varied widely from one another at 3% and 7% higher than the control, respectively. The latter was the highest CTE seen outside of the binary metakaolin concrete mixtures. MK1-15_SLa-30 saw the lowest CTE in the ternary group with a CTE of 9.73 × 10−6 mm/mm/°C (5.40 × 10−6 in/in/°F, an increase of 1% from control). MK2-15_SLb-30 saw the highest CTE in the ternary group with a CTE of 10.25 × 10−6 mm/mm/°C (5.70 × 10−6 in/in/°F, an increase of 7%). As a group the ternary SCM Infrastructures 2020, 5, 14 mixtures averaged a CTE of 9.93 × 10−6 mm/mm/°C (5.53 × 10−6 in/in/°F, an increase of 3%). 13 of 17 Figure 9. CTEs of binary benchmark and ternary SCM mixtures. 6. Summary, Discussion, and Recommendations 6.1. Summary This study evaluated the performance of ternary replacement mixtures. Specifically, a 45% replacement of cement with 15% metakaolin and 30% slag was evaluated for improved concrete performance including durability and dimensional stability. The study was conducted using a wide range of laboratory tests, and Table 4 provides a summary of binary SCM and ternary SCM performance. Table 4. Summary of alternate binary and ternary mixture performance compared to control (% change). Note: The up and down arrows indicate increases and decreases, respectively; arrows on both sides indicate insignificant change; and Pass (P)/Fail (F). Performance Category Mixture Properties Compression (28-day) Split-Cylinder Tension Mechanical Modulus or Rupture Dynamic Modulus (Ed ) RCPT Permeability Durability Sulfate Resistance ASR Resistance CTE Dimensional Stability Shrinkage Binary Ternary FA-45 SLa-30 SLb-30 MK1-15 _SLa-30 MK1-15 _SLb-30 MK2-15 _SLa-30 MK2-15 _SLb-30 MK3-15 _SLa-30 MK3-15 _SLb-30 ↓ ↓ ↑ ↑ ↓ ↓ ↓ ↔ ↓ −51% −2% 18% 10% −9% −8% −10% 0% −4% ↓ ↓ ↑ ↑ ↓ ↓ ↑ ↑ ↓ −24% −15% 17% 1% −4% −8% 31% 11% −4% ↓ ↑ ↑ ↔ ↓ ↓ ↓ ↓ ↓ −23% 4% 35% 0% −8% −21% −25% −15% −5% ↓ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ −28% 3% 1% −12% −22% −15% −15% −13% −13% ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ 42% 4% −48% −74% −83% −69% −85% −84% −85% - - - - - - - - - P F F P P P P P P - - - - - - - - - P F F P P P P P P ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ −2% 3% 7% 1% 4% 4% 7% 4% 5% ↑ ↔ ↓ ↓ ↓ ↓ ↓ ↓ ↓ 10% 0% −17% −28% −10% −21% −27% −28% −21% Infrastructures 2020, 5, 14 14 of 17 6.2. Discussion of Results 6.2.1. No Significant Reduction in Mechanical Strength Overall, mechanical strength of ternary mixtures were equal to or just below control. Only one mixture, MK1-15_SLa-30, managed to obtain a 28-day compressive strength higher (10%) than control. For comparison, the average compressive strength increase over control for all binary MK mixtures was 18% (ranged from -9% to 44%). This mixture also had the highest tensile strength of the ternary group, with a MOR equal to that of the control. Although the ternary mixtures out-performed binary concrete mixtures incorporating SLa, concrete mixtures incorporating SLb saw higher compressive (18% higher than control) and tensile strengths (MOR, 35% higher than control) than all ternary mixtures. Moreover, SLb-30 exhibited the highest tensile strength of all concrete mixtures tested. The early-age strength properties of the ternary mixtures appear to be primarily dependent on the slag product used, with the SLb concrete mixtures recording higher 1-day and 7-day strengths. Ternary mixtures incorporating SLa and SLb exhibit nearly the same strength as the binary SLa and SLb binary mixtures, respectively. The difference in compressive strength between concrete mixtures incorporating SLa and SLb continue to be visible up to 7 days (see Figure 2). After this, all ternary mixtures approach compressive strength of the control. Both MK and slag react pozzolanically with CH in ternary mixtures, which promotes optimum CH consumption and higher paste densities. This is evidenced by the durability results presented in this study. However, this does not necessarily ensure higher mechanical strength, as unreacted SCM is most likely present due to competing pozzolanic activity [37]. This explanation is supported by research that showed that as the level of MK inclusion increased, optimum compressive strength was found by reducing slag contents [14]. Although slag is also a latent hydraulic material, its CaO content is less than that of OPC, and thus less CH is produced during hydration when slag replaces a portion of cement. In short, in ternary mixtures incorporating 15% MK and 30% slag, the slight reduction in compressive strength is a result of less CH for pozzolanic activity, and more unreacted pozzolan. The increased density of the paste matrix, and additional C–S–H by means of pozzolanic reactions, compensate for the unreacted material. 6.2.2. Significantly Improved Durability The most substantial benefits of the ternary mixtures were seen by the tests of durability, which were most likely the result of high CH consumption. All ternary mixtures indicated very low permeabilities. Five total mixtures passed less than 500 Coulombs (C), and two ternary mixtures were less than 400 C. The ternary mixtures including SLb recorded the lowest permeability, and the highest when combined with MK3 (study low of 419 C). In addition, a sulfate expansion of less than 0.60% was achieved by only one binary mortar mixture (MK1-20). By contrast, five of six ternary mortars finished over the 6-month testing period under this value. Similarly, all six ternary mortars met ASR expansion criteria. This indicates that, although durability performance varies widely within binary slag and binary MK mortars (see Figures 5–7), ternary mixture durability is consistently high regardless of product combinations. 6.2.3. Slight Reduced Dimensional Stability All ternary concrete mixtures exhibited higher CTE values than the control, however, they were much less those of the binary MK mixtures [38]. As mentioned, increased porosities in FA and slag concrete mixtures have been correlated with decreases in paste CTEs [38]. This is a geometrical property. It is only when high CH consumption exists, that low-porosity pastes exhibit decreased CTEs from control. This is attributed to the high CTE of CH being replaced by lower CTE C–S–H now being the dominant effect. Figure 5 indicated that ternary concrete mixtures incorporating SLa have higher penetrability (and by inference, higher porosities) than SLb concrete mixtures on average. Figure 9 indicated that SLa concrete mixtures exhibit lower CTE values than SLb on average. It is Infrastructures 2020, 5, 14 15 of 17 hypothesized that the reduction in porosity lead to higher CTE values in SLb ternary blends. In ternary blends, the CTE is higher than control but significantly lower than binary MK concrete mixtures, which would most likely have lower porosities than ternary concrete mixtures. Overall, CTEs of ternary concrete mixtures were well below the 10 × 10−6 mm/mm/◦ C limit for acceptable performance in jointed concrete pavements [39] and other mass concrete elements [40]. 6.3. Recommendations for Usage Binary mixtures including MK show dramatic benefits in resistance to chemical attack and durability [38], however, ternary mixtures including metakaolin (MK) and slag (SL) also see these benefits. If mechanical strengths need only be similar to a control mixture (pertaining to ordinary Portland cement), it is recommended that a ternary mixture be used. Ternary mixtures see additional benefits above binary MK mixtures: • • • • Lower coefficient of thermal expansion (CTE) Lower heat of hydration [9,38] Increased resistance to chemical attack Reduced permeability It should be recognized that the durability of ternary mixtures is consistently high regardless of metakaolin and slag product combinations, whereas the performance of binary mixtures was product dependent [38]. 7. Conclusions The performance of ternary blended mixtures including metakaolin (MK) and blast furnace slag (SL) is reported through a wide range of laboratory tests. Major findings from this study are: • • • • • All concrete mixtures incorporating MK as a replacement of cement require a high dosage of superplasticizer (≥4 mL/kg of cementitious material, or 6.1 oz./cwt). Ternary concrete mixtures using MKs and slags (15% MK + 30% slag) tend to perform similarly to one another and control in both compression and tension at 28 days of age. Early-age strength characteristics of the ternary mixtures resemble the early-age strength characteristics of the 30% mixture using the same slag. Ternary concrete mixtures using MKs and slags exhibit very high resistance to chemical attack and very low chloride-ion penetrability. All combinations of commercially-available products performed similarly to one another. Ternary concrete mixtures using commercially-available metakaolin and slag products result in increased coefficient of thermal expansion (CTE) values, although much lower than binary MK CTEs previously studied. Values were near threshold value of 10 × 10−6 mm/mm/◦ C reported to be deleterious in jointed concrete pavements and other mass concrete elements. Drying shrinkage was lowered from control by ternary mixtures and was on average lower than the shrinkage of the binary MK mixtures. 8. Future Work A time history of the CTEs should be studied if ternary mixtures are to be used in mass concrete placements. A previous study [9] has reported the reduced heat of hydration in these ternary mixtures. The relationship between CTE and the extent of temperature-induced cracking should be of future interest. Finally, varying replacement levels and proportions of metakaolin and slag should be considered in future work. A microstructural analysis including SEM/EDS on mixtures is strongly recommended to investigate the observed interactions. Infrastructures 2020, 5, 14 16 of 17 Author Contributions: The authors confirm contribution to the paper as follows: methodology, M.S.S., M.G.C., S.A.D.; experimental work, M.S.S., M.G.C.; data analysis and paper writing, M.S.S., M.G.C., S.A.D. All authors have read and agreed to the published version of the manuscript. Funding: The study presented in this paper was conducted by the University of Georgia under the auspices of the Georgia Department of Transportation (GDOT) Research Projects 16-16 and 16-30 and thus funded by GDOT. Acknowledgments: The authors extend our sincere appreciation to GDOT staff and engineers that supported this research. Special thanks to David Jared, Peter Wu, and Gary Wood for their continuous support. The authors greatly appreciate generous material donations made by BASF, ACT, and Thiele/Burgess Pigment as well as Argos and Lehigh Hanson. The funding sponsors had no role in the design, analysis, or interpretation of data. The opinions, findings, and conclusions may not reflect the views of the funding agency or other individuals. Conflicts of Interest: The authors have no implicit or explicit conflict of interest of any kind in this study. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Kosmatka, S.H.; Wilson, M.L. Design and Control of Concrete Mixtures, 15th ed.; Portland Cement Association: Skokie, IL, USA, 2011. Gibbs, M.J.; Soyka, P.; Conneely, D.; Kruger, M. CO2 Emissions from Cement Production. 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https://openalex.org/W4243192585	https://www.ajol.info/index.php/wsa/article/download/180593/169943	English	null	The commodity systems of <i>Brassica rapa</i> L. subsp. chinensis and <i>Solanum retroflexum</i> Dun. in Vhembe, Limpopo Province, South Africa	Water S.A./Water SA	2,018	cc-by	6,331	Introduction rum (S. americanum), S. nigrum and S. scabrum (Edmonds and Chweya, 1997). S. retroflexum is an annual branching herb that grows up to 75 cm in height, has alternate, heavily veined leaves with small hairs on both sides, white flowers with a yellow cen- tre and green berries that turn dark purple when ripe (Grabandt, 1985; Schippers, 2002). In this study, the smallholder commodity systems of Brassica rapa L. subsp. chinensis and Solanum retroflexum Dun. are described and analysed. In South Africa, these two vegetables are exclusively produced and consumed by African people. Relative to white cabbage (B. oleracea L. var. capitata), which is arguably the most commonly produced and consumed leafy vegetable among black people in South Africa, the two crops studied here are richer in nutrients per unit fresh mass (Table 1). This makes them potentially suitable elements for inclusion in food-based approaches aimed at combating hidden hunger, which is an important type of under-nutrition among poor rural people in South Africa. B. rapa subsp. chinensis refers to non-heading types of Chi- nese cabbage (Opeňa et al., 1988), an annual, flowering vegetable with dark green leaves supported by light green to white petioles that form a rosette (Hill, 1990). Chinese cabbage has a short grow- ing season. From sowing to the end of the vegetative stage takes about six weeks for early maturing cultivars and 11 weeks for late maturing cultivars (Rubatzky andYamaguchi, 1997; Hong- Fu, 1988). Depending on the cultivar, the plants reach a height of 15 cm to 30 cm at the end of the vegetative stage. The progenitor form of Chinese cabbage, Brassica campestris, is believed to have evolved in the Mediterranean from where it was introduced to China more than 2000 years ago and dif- ferentiated into the various sub- species (Opeňa et al., 1988). The crop probably found its way from Asia into Africa as a result of trade between these two continents. l Calcium, iron and B Brassica oleracea L Solanum nigrum (adap Scientific name Brassica. oleracea L. var. Brassica rapa L. subsp. ch TABLE 1 Calcium, iron and Beta-carotene content of 100 g fresh edible portion of Brassica oleracea L. var. capitata, Brassica rapa L. subsp. chinensis and Solanum nigrum (adapted from Opeňa et al., 1988; Edmonds & Chweya, 1997) Scientific name Common Eng- lish name Calcium Iron Beta-carotene equivalent (g) (mg) (µg) Brassica. oleracea L. var. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) Abstract Using farmer surveys the smallholder commodity systems of Brassica rapa L. subsp. chinensis and Solanum retroflexum Dun. in Vhembe District, Limpopo Province, South Africa were described and analysed. Production, transaction and con- sumption of the two vegetables were deeply embedded in the food, trade and farming systems of local people. The cultivation systems, which appeared effective, were developed by borrowing elements from the systems of other crops to which new knowledge specific to B. chinensis and S. retroflexum were added. Several elements of the production systems that could benefit from scientific enquiry were identified. These included genetic improvement and the optimisation of planting density and nutrient supply. Keywords: Smallholder commodity systems, filière, African leafy vegetables; Chinese cabbage, nightshade The commodity systems of Brassica rapa L. subsp. chinensis and Solanum retroflexum Dun. in Vhembe, Limpopo Province, South Africa# W van Averbeke, TE Tshikalange and KA Juma Centre for Organic and Smallholder Agriculture, Department of Crop Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa W van Averbeke, TE Tshikalange and KA Juma Centre for Organic and Smallholder Agriculture, Department of Crop Sciences, Tshwane University of Technolog Private Bag X680, Pretoria 0001, South Africa # Revised version. Originally presented at the International Sympo- sium on the Nutritional Value and Water Use of Indigenous Crops for Improved Livelihoods held on 19 and 20 September 2006 at the University of Pretoria in Pretoria, South Africa  +2712 382 5777; fax: +2712 382 5869; # Revised version. Originally presented at the International Sympo- sium on the Nutritional Value and Water Use of Indigenous Crops for Improved Livelihoods held on 19 and 20 September 2006 at the University of Pretoria in Pretoria, South Africa * To whom all correspondence should be addressed.  +2712 382 5777; fax: +2712 382 5869; e-mail: vanaverbekew@tut.ac.za * To whom all correspondence should be addressed.  +2712 382 5777; fax: +2712 382 58 e-mail: vanaverbekew@tut.ac.za Introduction capitata White cabbage 55 0.8 280 Brassica rapa L. subsp. chinensis Non-heading Chinese cabbage 102 2.6 2305 Solanum nigrum L. Black night- shade 266 2.6 6650 e-mail: vanaverbekew@tut.ac.za Consumption holder commodity chains or systems. sible participants were requested to demonstrate their practices. This enabled the collection of quantitative data on inter alia fer- tiliser application rates and plant spacing, using a portable scale and a measuring tape to make the measurements. Purposive sampling (Strydom, 2005), which is a non-probability sampling method, was used to compose the sample of participants. This involved the identification of farmers who actively produced the crops under consideration, using the resident extension officers and other farmers as sources of referral. Forty irrigation farm- ers participated in the B. chinensis commodity system survey, namely seven from Tshiombo (22o48’S; 30o30’30”E), four from Murara (22o53’S; 30o30’30”E), two from Palmaryville (22o59’S; 30o26’E), 16 from Khumbe (23o03’S; 30o21’E), five from Rab- ali (22o53’S; 30o07’E) and six from Rammbuda (22o59’30”S; 30o25’30”E). In total 50 irrigation farmers participated in the S. retroflexum commodity system survey, namely 30 at Dzindi (30°23’S; 23°01’E), 9 at Rabali and 11 at Khumbe. sible participants were requested to demonstrate their practices. This enabled the collection of quantitative data on inter alia fer- tiliser application rates and plant spacing, using a portable scale and a measuring tape to make the measurements. Purposive sampling (Strydom, 2005), which is a non-probability sampling method, was used to compose the sample of participants. This involved the identification of farmers who actively produced the crops under consideration, using the resident extension officers and other farmers as sources of referral. Forty irrigation farm- ers participated in the B. chinensis commodity system survey, namely seven from Tshiombo (22o48’S; 30o30’30”E), four from Murara (22o53’S; 30o30’30”E), two from Palmaryville (22o59’S; 30o26’E), 16 from Khumbe (23o03’S; 30o21’E), five from Rab- ali (22o53’S; 30o07’E) and six from Rammbuda (22o59’30”S; 30o25’30”E). In total 50 irrigation farmers participated in the S. retroflexum commodity system survey, namely 30 at Dzindi (30°23’S; 23°01’E), 9 at Rabali and 11 at Khumbe. In the triple-A framework actors refer to the people or agen- cies involved in smallholder commodity chains, activities to what they do and artefacts to what they use in the conduct of their activities. These three elements constitute the core of the indigenous knowledge system that is applied in smallholder commodity chains. Indigenous knowledge resides within the actors. Through experimentation actors develop, adopt or adapt material and social technology to suit their circumstances. Tech- nology can be called an artefact because it is a human creation. Materials and methods Using the Triple-A framework as a guide, empirical data were collected by means of farmer surveys at selected sites in the Vhembe District. Vhembe is the most northern district of the Limpopo Province and borders Zimbabwe. This District is regarded as the origin of cultivation of B. rapa subsp. chinen- sis and S. retroflexum in South Africa. Vhembe forms part of the summer rainfall region and since both crops are grown dur- ing winter they are produced under irrigation. For this reason the surveys were conducted on smallholder irrigation schemes where the two crops are being produced. All of these smallholder projects were canal irrigation schemes with plot sizes that ranged between 0.6 ha and 1.3 ha. Face-to-face interviews guided by a checklist of issues were used to collect the data and where pos- Consumption Usually artefacts are material objects, but for the purpose of the study of smallholder commodity chains the concept can be expanded to include both tangible and intangible creations of people. These can include, for example, different landraces of a particular crop, equipment, materials and techniques used in the cultivation, harvesting or processing of the crop and the social arrangements that characterise transactions. The data were analysed qualitatively. This consisted of writ- ing up each interview in full, followed by analysing the informa- tion in these texts in accordance with the pre-determined themes contained in the checklist. Analysis of the different farmer practices paid particular attention to the degree of diversity or variability. When a particular practice showed limited vari- ability among farmers, this was interpreted as an indication of consensus among participants that this particular way of doing things consistently produced the best results. Conversely, when a practice was subject to wide diversity, this was interpreted as evidence of uncertainty among farmers of what worked best. Where information was of a quantitative nature, the data were converted to a standardised format. This enabled the determi- nation of measures of their central tendency and spread using the Descriptive Statistics routine of the Data Analysis routine contained in the Tools routine of MS Office Excel version 2003. The main focus of the analysis of the two commodity sys- tems was on the cultivation of the two vegetables. Analysis of the technical content of the existing cultivation system of a crop is important when seeking to contribute to its development through agronomic research. Existing systems contain elements of best-practice, which farmers have discovered and refined through observation and experimentation over many years. Figure 2 The Triple-A framework for the analysis of rural people’s knowledge related to food commodities (Van Averbeke and Khosa, 2004) Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) TABLE 1 TABLE 1 Calcium, iron and Beta-carotene content of 100 g fresh edible portion of Brassica oleracea L. var. capitata, Brassica rapa L. subsp. chinensis and Solanum nigrum (adapted from Opeňa et al., 1988; Edmonds & Chweya, 1997) Solanum retroflexu Dun. forms part of the S. nigrum com- plex of species and is indigenous to South Africa (Grabandt, 1985; Edmonds and Chweya, 1997). Other species in the Solanum nigrum complex that are com- monly found in Africa include S. gracile, S. villosum, S. nodiflo- Solanum nigrum L. A commodity system or chain, called filière in the French language, involves many activities, including production, col- lection, up-stream storage, transformation, down-stream stor- age, redistribution and consumption. The filière approach, illus- trated in Fig. 1, is used as a framework to investigate the way in which particular agricultural commodities are produced, stored, transformed, transacted and consumed in a locality or region (Leplaideur, 1994; Moustier et al., 1997). Van Averbeke and Khosa (2004) developed the Triple-A framework, illustrated in Fig. 2, to guide the study of small- 349 Production Collection Up-stream storage Down-stream storage Consumption Transformation Redistribution Production Transformation Consumption Actors Activities Artifacts Collection & Storage Storage & Redistribution Figure 1 Analytical framework used in filière studies (after Leplaideur, 1994) Figure 2 The Triple-A framework for the analysis of rural people’s knowledge related to food commodities (Van Averbeke and Khosa, 2004) Figure 1 Analytical framework used in filière studies (after Leplaideur, 1994) Down-stream storage Consumption Up-stream storage Figure 2 The Triple-A framework for the analysis of rural people’s knowledge related to food commodities (Van Averbeke and Khosa, 2004) Actors Activities Artifacts Figure 1 Analytical framework used in filière studies (after Leplaideur, 1994) Origin, diversity and reproduction Farmers mostly used chemical fertilisers super-phosphate (10.5% P), limestone ammonium nitrate (LAN 28% N), or urea (46% N), or the fertiliser mixtures 2:3:4 (30) and 2:3:2 (22) to supply nutrients to the crop, but some applied organic fertilisers, mostly poultry manure. Irrespective of the type of fertiliser used, farmers employed a similar nutri- ent supply strategy, which consisted of the application of organic manure or a chemical fertiliser mixture at planting, followed by one or more topdressings with a nitrogenous fertiliser, most often LAN. Farmers employed a diversity of methods to apply fertilis- ers, including broadcast, band and spot application. The rates at which N, P and K were applied also varied widely. Application rates of N ranged from 134 kg N·ha-1 to 545 kg N·ha-1 with a mean of 229 kg N·ha-1 and a median of 209 kg N·ha-1. Application rates of P ranged from 44 kg N·ha-1 to 277 kg P·ha-1 with a mean of 128 kg P·ha-1 and a median of 119 kg P·ha-1. Application rates of K ranged from 35 kg K·ha-1 to 207 kg K·ha-1 with a mean of 104 kg K·ha-1 and a median of 91 kg K·ha-1. On all the schemes included in the study, water was applied by means of short furrow irriga- tion (De Lange, 1994). Farmers pointed out that for optimum production the crop needed two or three irrigation per week, but in many instances the frequency of irrigation was limited to once per week because of the institutional arrangements gov- erning access to water limited farmers to this particular irriga- tion frequency(Letsoalo and Van Averbeke, 2006). Crop protec- tion was by chemical means. Respondents listed Bagrada bugs (Bagrada hilaris), cutworms (Agrotis segetum), cabbage aphids (Brevicoryne brassicae), spider mites (Tetranchus spp.), small red ants, birds and locusts as the main pests of Chinese cabbage. Commonly used chemicals for the control of insect pests were Metasystox (oxydemeton-methyl), Tamaron (methamidophos) and Cymbush (cypermethrin). Weeds were controlled by hand hoeing or by pulling out the weeds. During the growing season 2 to 8 weed control operations were performed.l i Before S. retroflexum was taken into cultivation, people in Vhembe harvested the plant from the wild for use as a vegetable. Respondents reported that in the wild, the plant was typically found under large trees in mountain forests. Birds were identi- fied as the most likely distributors of S. Origin, diversity and reproduction retroflexum in the wild, because they consumed the berries and probably excreted the seeds whilst resting on trees. The harvesting of S. nigrum spe- cies from the wild for use as a leafy vegetable is not limited to the Vhembe District. Similar use of S. scabrum has been reported in the KwaZulu-Natal and Eastern Cape Provinces of South Africa (Juma, 2006). However, cultivation of S. nigrum species in South Africa is at this stage restricted to the Vhembe District, where reportedly it commenced during the 1960s at Dzindi, the centre of indigenous knowledge of this crop (Juma, 2006). Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) Origin, diversity and reproduction Participants in the survey identified 11 different landraces of B. chinensis, all of which had been given local names. The Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) 350 landraces dabadaba and lidzhainthi were by far the most com- monly grown, followed by tshikete and mutshaina wa u navha. The other seven landraces were produced by a few farmers only. Participants named Zimbabwe as the origin of 10 of the 11 lan- draces that were identified. They said that petty traders from Zimbabwe, who marketed bambara groundnuts in Vhembe, had been distributing the seed of the different landraces of Chinese cabbage. Dabadaba was the only exception. Some participants claimed that this landrace was transferred from the wild by local people, whilst others believed that it also entered Vhembe from Zimbabwe. Participants identified the village of Maraxwe, near the Tshiombo and Rammbuda Irrigation Schemes, as the cen- tre of indigenous knowledge of Chinese cabbage in Vhembe. Indications are that cultivation of this crop commenced during the 1940s. Once farmers had acquired seed of a particular lan- drace they sustained its reproduction. Utilisation of the different landraces was spread among farmers through seed exchanges. Efforts by farmers to improve the crop were rare and limited to selecting seed from plants that produced exceptionally large numbers of leaves, which was done by three of the 40 partici- pants. The others left their entire stand to mature and dry on the land and then collected the dry plants for threshing and seed collection. Threshed seed was stored in different types of con- tainers, including glass bottles, plastic containers, paper, plastic, woven polypropylene bags, pieces of cloth and dzikhali, which are traditional clay pots that are covered with a clay lid. Seeds stored in dzikhali were reported to maintain their viability the longest, up to three years, whilst seed stored in other containers only lasted up to two years. The extent to which this is true was not verified. which was then covered with a thin layer of about 5 to 7 mm soil. Watering cans or perforated buckets were used to irrigate the soil of the seedbed, and this was done every second day or more frequently. Origin, diversity and reproduction Seedlings typically emerged 2 to 7 d after sowing and were transplanted 3 to 6 weeks after sowing, when they had reached the 3rd to 6th leaf stage. When the seedlings were ready for transplanting, the seedbed was first watered thoroughly and then the transplants were pulled out from the seed bed by hand or dug out using a flat stick. Before being planted to the crop, the irrigation plot was ploughed, disked and ridged using mechani- cal means and then watered. Planting of seedlings was done by placing the roots of the transplants in shallow planting holes of about 3 cm deep, made by means of a round stick, followed by covering the roots with soil. The final planting density typically ranged between 4 and 8 plants·m-2. Most respondents planted Chinese cabbage during April and May. They pointed out that earlier planting had the advantage of superior market conditions, but the disadvantage that it increased the incidence of pests. All participants applied fertilisers to increase the growth and yield of the Chinese cabbage plants. Farmers mostly used chemical fertilisers super-phosphate (10.5% P), limestone ammonium nitrate (LAN 28% N), or urea (46% N), or the fertiliser mixtures 2:3:4 (30) and 2:3:2 (22) to supply nutrients to the crop, but some applied organic fertilisers, mostly poultry manure. Irrespective of the type of fertiliser used, farmers employed a similar nutri- ent supply strategy, which consisted of the application of organic manure or a chemical fertiliser mixture at planting, followed by one or more topdressings with a nitrogenous fertiliser, most often LAN. Farmers employed a diversity of methods to apply fertilis- ers, including broadcast, band and spot application. The rates at which N, P and K were applied also varied widely. Application rates of N ranged from 134 kg N·ha-1 to 545 kg N·ha-1 with a mean of 229 kg N·ha-1 and a median of 209 kg N·ha-1. Application rates of P ranged from 44 kg N·ha-1 to 277 kg P·ha-1 with a mean of 128 kg P·ha-1 and a median of 119 kg P·ha-1. Application rates of K ranged from 35 kg K·ha-1 to 207 kg K·ha-1 with a mean of 104 kg K·ha-1 and a median of 91 kg K·ha-1. On all the schemes included in the study, water was applied by means of short furrow irriga- tion (De Lange, 1994). Origin, diversity and reproduction Farmers pointed out that for optimum production the crop needed two or three irrigation per week, but in many instances the frequency of irrigation was limited to once per week because of the institutional arrangements gov- erning access to water limited farmers to this particular irriga- tion frequency(Letsoalo and Van Averbeke, 2006). Crop protec- tion was by chemical means. Respondents listed Bagrada bugs (Bagrada hilaris), cutworms (Agrotis segetum), cabbage aphids (Brevicoryne brassicae), spider mites (Tetranchus spp.), small red ants, birds and locusts as the main pests of Chinese cabbage. Commonly used chemicals for the control of insect pests were Metasystox (oxydemeton-methyl), Tamaron (methamidophos) and Cymbush (cypermethrin). Weeds were controlled by hand hoeing or by pulling out the weeds. During the growing season 2 to 8 weed control operations were performed. S. retroflexum was grown from seed and reproduction was entirely maintained by local farmers. All farmers followed which was then covered with a thin layer of about 5 to 7 mm soil. Watering cans or perforated buckets were used to irrigate the soil of the seedbed, and this was done every second day or more frequently. Seedlings typically emerged 2 to 7 d after sowing and were transplanted 3 to 6 weeks after sowing, when they had reached the 3rd to 6th leaf stage. When the seedlings were ready for transplanting, the seedbed was first watered thoroughly and then the transplants were pulled out from the seed bed by hand or dug out using a flat stick. Before being planted to the crop, the irrigation plot was ploughed, disked and ridged using mechani- cal means and then watered. Planting of seedlings was done by placing the roots of the transplants in shallow planting holes of about 3 cm deep, made by means of a round stick, followed by covering the roots with soil. The final planting density typically ranged between 4 and 8 plants·m-2. Most respondents planted Chinese cabbage during April and May. They pointed out that earlier planting had the advantage of superior market conditions, but the disadvantage that it increased the incidence of pests. All participants applied fertilisers to increase the growth and yield of the Chinese cabbage plants. Cultivation practices Fresh Chinese cabbage leaves, to which cut-up tomatoes are added, are boiled until soft, squeezed by hand into small balls of about 2 to 4 cm in diameter, pressed down on a zinc roof sheet to yield a patty shape, and left to sun-dry. The dry patties are stored in plastic containers and consumed or sold during summer, when fresh B. chinensis is not available. Another preservation method involves the storing of blanched leaves without adding spices in a container in a freezer. Stored in this way the vegetable is said to retain its colour and taste for up to a year. Both fresh and proc- essed leaves are used to prepare a relish to accompany maize porridge. There are two main ways of preparing the relish. One way is to stew Chinese cabbage leaves in a little water to which salt, tomato and cooking oil were added to improve the taste. The other involves the addition of peanut meal, resulting in a relish called dovhi. The flowers of the dabadaba landrace and the leaves attached to its peduncle are also used as a medicine to treat high blood pressure.li S. retroflexum is harvested for the first time about four to eight weeks after transplanting. This involves the cutting off the top branches of the plant, whilst leaving two or three branches at the base of plant. The majority of farmers commence harvesting when the branches of the plants have spread sufficiently to cover the ridges separating the irrigation furrows. They harvest the crop for the second time when the shoots have re-grown sufficiently, which usually occurs about 2 to 3 weeks after the first harvest. Harvesting ends when the crop starts to produce narrow, thin leaves, because these are no longer marketable. Fruit formation is also used as an indicator that it is time to stop the harvesting of shoots, because fruit formation is said to adversely affect the qual- ity of the leaves, turning them sour and leathery. No evidence of processing of leaves was found. As with Chinese cabbage, petty traders controll the S. retroflexum market, but since 2005 the crop has also been on offer on the shelves of local stores that form part of a major national supermarket chain, being supplied daily and directly by selected farmers who owned trucks and special- ised in the production of this crop. Cultivation practices Application rates ranged from 22 kg N·ha-1 to 547 kg N·ha-1, with a mean of 208 kg N·ha-1 and a median of 185 kg N·ha-1. Phosphorus application rates used by S. retroflexum cultivators ranged from 6 kg P·ha-1 to 295 kg P ha-1, with a mean of 51 kg P·ha-1 and a median of 42 kg P·ha-1. The application rates of potassium ranged from 4 kg K·ha-1 to 392 kg K·ha-1, with a mean of 47 kg K·ha-1 and a median of 34 kg K·ha-1. In all three schemes farmers were institutionally restricted to a single irrigation per week. Generally, respondents identified lack of water for irrigation as a constraint in the cultivation of S. retroflexum, suggesting that irrigation frequencies higher than once per week are needed for optimum growth of the crop. H t k ti i d Typically, the 5th leaf has a length of about 25 cm and a width of about 15 cm. Farmers regarded the first four leaves to be too small to be marketable and many farmers pulled the first four leaves off the plant and discarded them when harvesting the 5th leaf, because they believed that this stimulated growth. About one week after harvesting the 5th leaf, subsequent leaves were harvested. The number of marketable leaves produced by Chi- nese cabbage depends on the landrace and the time of plant- ing. The dabadaba landrace typically produces between 10 and 16 leaves, of which 6 to 12 leaves are large enough to be marketable. Harvesting the leaves of a plant continues until the peduncle has elongated and the first flowers open. Marketing of the crop is mainly done by petty traders who visit the irrigation schemes to purchase vegetables for sale on the streets of local towns. Small quantities are also sold by farmers to people in the villages surrounding the Schemes. Since 2005, supermarkets in the towns of Vhembe retail B. chinensis but none of the farm- ers covered by the survey used this particular channel to market the crop. About one in 10 farmers processes Chinese cabbage to avoid losses when part of the produce failed to find a market, which affected the June and July plantings in particular. Cultivation practices The seed of B. chinensis is 1.5 to 2 mm in size and fairly dense, making it possible to practise direct seeding, which was done by 33% of participants. When using direct seeding farmers opened a shallow planting furrow of about 2 to 3 cm deep using a hand hoe, sprinkled the seed in the furrow, covered the seed with about 1 to 2 cm of soil, and then irrigated the soil. Once the plants had emerged and had 3 to 5 true leaves, they were thinned out to achieve the desired stand. Often the plants that were pulled out during thinning were used to plant additional land to Chinese cabbage. Farmers who planted their plots using transplants pro- duced these in specially prepared seed beds. These consisted of small areas in which the soil was worked to a fine tilth using a garden fork or a hand hoe. After incorporating fertilisers into the soil of the seedbed farmers used a stick or finger to open shallow (about 10 mm deep) and narrow (about 15 to 20 mm wide) furrows spaced 3 to 5 cm apart. In these furrows they sprinkled the seed, S. retroflexum was grown from seed and reproduction was entirely maintained by local farmers. All farmers followed the same procedure to harvest the seed. The ripe fruit of the crop was collected, soaked in a container filled with water and then squeezed by hand whilst still in the water to separate the seed from the rest of the fruit. Decanting the supernatant and refilling the bucket with fresh water was sustained until the water remained clear. After the final decant, the seed which is denser than water was transferred onto plastic sheets or dis- carded polypropylene grain bags to dry. Once dry, the seed was stored in dry pieces of cloth or in sealed containers made from 351 glass or plastic. Farmers pointed out that all of these storage methods ensured that the seed remained viable for at least two years. Seed selection, being one of the ways to increase pro- duction, was not given much attention by farmers. Only two of the 30 respondents at Dzindi selected specific plants for seed. One collected large fruit from plants with broad leaves, whilst the other set aside a group of plants specifically for seed pro- duction and did not harvest the shoots of these plants. Cultivation practices Being small (0.5 to 1.5 mm), the seed of S. retroflexum is not well suited for direct seeding, explaining why all respondents made use of transplants. These were produced in exactly the same way as described for B. chinensis. The duration of germination and emergence varied widely, ranging between five and 28 d. According to Schippers (2002), the duration of germination of S. nigrum species is determined by the degree to which sug- ars and germination inhibitors present in the fruit have been removed from the seed. Transplanting of the seedlings was done 3 to 4 weeks after emergence. Farmers considered colour and degree of development of the seedlings when deciding on when to transplant. Generally, seedlings were considered ready for transplanting when their colour had turned from pale-green to deep green and when they had developed about 5 to 6 true leaves. To remove the transplants from the seedbed the seed- lings were carefully uprooted. Soil attached to the roots of the seedlings was washed off. Farmers claimed that this enhanced root development of the seedlings. The seedlings were placed in holes of about 3 to 5 cm deep, which were drilled in the soil using a stick or forefinger. A single seedling was placed in each hole and then the soil around the roots was pressed down to ensure good contact between roots and soil. Whilst the major- ity of farmers practiced single row planting, some planted in double rows, which reportedly was done to suppress weed com- petition. The spacing between the plants varied widely among producers. Inter-row spacing ranged between 0.32 m to 0.90 m with a mean of 0.75 m and intra-row spacing from 0.12 to 0.60 m with a mean of 0.29 m. Planting density varied widely, ranging between 1.9 plants·m-2 and 26.0 plants·m-2, with a mean of 6.3 plants·m-2. The majority of respondents (55%) used a planting density ranging between 3 and 5 plants·m-2. Farmers employing planting densities in excess of 9 plants·m-2 (19%) all achieved these by using a double-row or tram-line planting pattern. The majority of farmers (98%) used chemical fertilisers, but 36% also applied organic fertilisers. Only one respondent applied organic fertilisers only. The nutrient supply strategy used by S. retroflexum producers was the same as that described for Chi- nese cabbage and the methods of applying fertilisers displayed the same type of diversity. Cultivation practices This development is a clear indication that the S. retroflexum commodity system is expand- ing in Vhembe. In the study area, leaves and young shoots of S. retroflexum are cooked and used as a relish. These plant parts are first separated from the main stems and then boiled in water. Sunflower oil, tomatoes and salt are added to enhance flavour. During cooking the first boiling water is discarded to reduce bitterness. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) References DE LANGE M (1994) Small Scale Irrigation in South Africa. Water Research Commission, Rietfontein, Pretoria. 29 pp. EDMONDS JM and CHWEYA JA (1997) Black nightshades: Solanum nigrum L. and related species: promoting the conservation and use of underutilised and neglected crops. IPGRI, Rome. [Online]. Avail- able from: http://www.bioversityinternational.org/publications/ pufile.asp?_pub=337 (Accessed on22/03/07). GRABANDT K (1985) Weeds of Crops and Gardens in South Africa. Ciba-Geigy (Pty) Ltd, Johannesburg. 134 pp. HILL DE (1990) Chinese cabbage and pak choi trials 1988-1989. Bul- letin 879. The Connecticut Agricultural Experiment Station, New Haven. 11 pp. There were indications that agronomic research could bring about improvements to the existing systems. Specific elements of the production systems that could benefit from scientific enquiry were identified. These elements included genetic improvement of the crops, the effect of planting date on rate of development and yield for use in market planning and determining the water requirements of the two crops for optimum production. Using variability as an indicator of farmer uncertainty about what constituted best practice, nutrient supply was identified for both crops as one of the practices that needed research attention. The wide variability in nutrient application rates used by producers of both crops suggests that a significant proportion of farm- ers may be applying either too little or too much fertilisers for optimum production. Determining optimum planting density in S. retroflexum is another issue for research from which farmers are likely to benefit. HONG-FU Y (1988) The effect of nitrogen fertilizer application on the yield of Chinese cabbage. Asian Vegetable Research and Develop- ment Center Report [Online]. Available from: http://www.arc-avrdc.i org/pdf_files/Yanghongfu(6-N).pdf. (Accessed on 11/03/05) JUMA KA (2006) Response of Solanum retroflexum Dun. to nitrogen, phosphorus and potassium in pots. M. Tech. (Agric.) Dissertation, Department of Crop Sciences, Tshwane University of Technology, Pretoria. 138 pp. LEPLAIDEUR A (1994) Rencontre de la petite production marchande et du commerce informel des vivres en PVD: méthode d’analyse des rapports sociauxs. In: Benz H, Lançon F, Leplaideur A, Moustier P and Pujo L (eds.) Méthodes d’analyse des rapports sociaux dans les échanges vivriers en Afrique et en Asie du Sud. Documents de travail du CIRAD-CA No 5-94. Centre de coopération internation- ale en recherche agronomique pour le developpement, Montpellier. 7-19. LETSOALO SS and VAN AVERBEKE W (2006) Water management on a smallholder canal irrigation scheme in South Africa. Harvest, marketing, processing and use The 5th true leaf of Chinese cabbage was the first that was con- sidered suitable for harvest and consumption. This particular leaf was harvested when the plants had reached the 8-leaf stage. 352 authors accept full liability for any opinions, findings, conclu- sions or recommendations contained in this article. Conclusions The description of the commodity systems of B. rapa subsp. chinensis and S. retroflexum in Vhembe showed that production, transaction and consumption of the two vegetables are embed- ded in the food, trade and farming systems of local people. For both crops farmers had developed cultivation systems that appeared to be effective. They developed these systems by bor- rowing elements from the systems they used for the production of other crops to which they had added new knowledge specific to the crops concerned. This new knowledge had been acquired by means of on-farm experimentation and farmer-to-farmer knowledge exchanges and applied specifically to the collection of seed and the method of harvesting the leaves of the crops. References In: Perret S, Farolfi S and Hassan R (eds.) Water Governance for Sustainable Development: Approaches and Lessons from Developing Countries. Earthscan, London. 93-110. The local market for the two crops showed signs of expan- sion, but this did not prevent production from exceeding demand, particularly in the case of Chinese cabbage. Marketing of the two crops is primarily in the hands of petty traders who retail the crops in the streets of local towns. The high rate of migration of rural people to South Africa’s metropolitan areas during the past two decades suggests the possibility of important but dis- tant metropolitan markets for the two crops, but these still need to be explored.l MOUSTIER P, BERTON S and SECK PA (1997) Filières maraîchères en Afrique: guide pratique d’analyse. Rapport FAO/CGP/RAF/244 version provisoire. CIRAD, Montpellier. 105 pp. Ň OPEŇA RT, KUO CG and YOON JY (1988) Breeding and seed pro- duction of Chinese cabbage in the tropics and subtropics. Technical Bulletin No 17. Shanshua, Taiwan: Asian Vegetable Research and Development Center (AVRDC). 92 pp. The local way of preparing S. retroflexum involves the dis- carding of the first boiling water, a practice known to lessen the nutrient content of vegetables (Schippers, 2002). In the East- ern Cape, S. scabrum is boiled in combination with other leafy vegetables collected from the wild, including Sonchus asper, Chenopodium album and Amaranthus thunbergii. This reduces the bitter taste of the relish and obviates the need to discard the first boiling water (Siwundla, 2006). Similar approaches could be introduced in Vhembe to ensure that the nutrients contained in S. retroflexum are retained when the vegetable is prepared for consumption. RUBATZKY VE and YAMAGUCHI M (1997) World Vegetables: Prin- ciples and Nutritive Values. (2nd edn.) Chapman and Hall, New York. 843 pp. SCHIPPERS RR (2002) African Indigenous Vegetables: An Overview of the Cultivated Species. Revised edition. [CD-ROM]. Natural Resources Institute/ACP-EU Technical Center for Agricultural and Rural Cooperation, Chatham. 252 pp. p pp STRYDOM H (2005) Sampling and sampling methods. In: De Vos AS, Strydom H, Fouché CB and Delport CSL (eds.) Research at Grass Roots (3rd edn.) Van Schaik, Pretoria. 192-204. Strydom H, Fouché CB and Delport CSL (eds.) Research at Grass Roots (3rd edn.) Van Schaik, Pretoria. 192-204. ( ) SIWUNDLA N (2006) Personal communication from Ms Nontsikelelo Siwundla who grew up on a farm near Debe Nek in the Eastern Cape. 27 August. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) Acknowledgement VAN AVERBEKE W and KHOSA TB (2004) The Triple-A framework for the analysis of smallholder food commodity chains. In: Proc. 3rd Int. Conf. Entrepreneurship – Sustainable Globalization, 3-4 November, Pretoria. [CD-ROM]. Tshwane University of Technol- ogy, Pretoria, South Africa. 292-299. This article is based on work done in terms of a research project that is being supported financially by the Tshwane University of Technology, the South African Water Research Commission (WRC Project No. K5/1464//4 and WRC Project No. K5/1579//4) and the National Research Foundation (GUN2069585) but the Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 33 No. 3 (Special Edition) 2007 ISSN 1816-7950 = Water SA (on-line) 353 353 354
https://openalex.org/W2559260541	https://hal.archives-ouvertes.fr/hal-01604527/file/Dubois_2016_Plos%20pathogens_1.pdf	English	null	Correction: Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis	PLOS pathogens	2,016	cc-by	1,615	Correction: necrotrophism Is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis Thomas Dubois, Karoline Faegri, Sébastien Gélis-Jeanvoine, Stéphane Perchat, Christelle Lemy, Christophe Buisson, Christina Nielsen-Leroux, Michel Gohar, Philippe Jacques, Nalini Rama Rao, et al. To cite this version: Thomas Dubois, Karoline Faegri, Sébastien Gélis-Jeanvoine, Stéphane Perchat, Christelle Lemy, Christophe Buisson, Christina Nielsen-Leroux, Michel Gohar, Philippe Jacques, Nalini Rama Rao, et al. To cite this version: Thomas Dubois, Karoline Faegri, Sébastien Gélis-Jeanvoine, Stéphane Perchat, Christelle Lemy, et al.. Correction: necrotrophism Is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis. PLoS Pathogens, 2016, 12 (11), pp.e1006049. 10.1371/journal.ppat.1006049. hal-01604527 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01604527 https://hal.science/hal-01604527v1 Submitted on 27 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. Distributed under a Creative Commons Attribution 4.0 International License OPEN ACCESS Citation: Dubois T, Faegri K, Ge´lis-Jeanvoine S, Perchat S, Lemy C, Buisson C, et al. (2016) Correction: Necrotrophism Is a Quorum-Sensing- Regulated Lifestyle in Bacillus thuringiensis. PLoS Pathog 12(11): e1006049. doi:10.1371/journal. ppat.1006049 The conclusion that lipopeptide kurstakin is necessary for the survival of the wild type Bt strain 407 Cry- in the insect cadavers is therefore incorrect. The conclusions of this publication that remain valid are as follows: 1. During infection of an insect host, the quorum sensor NprR is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. CORRECTION Correction: Necrotrophism Is a Quorum- Sensing-Regulated Lifestyle in Bacillus thuringiensis a1111 OPEN ACCESS Citation: Dubois T, Faegri K, Ge´lis-Jeanvoine S, Perchat S, Lemy C, Buisson C, et al. (2016) Correction: Necrotrophism Is a Quorum-Sensing- Regulated Lifestyle in Bacillus thuringiensis. PLoS Pathog 12(11): e1006049. doi:10.1371/journal. ppat.1006049 CORRECTION Correction: Necrotrophism Is a Quorum- Sensing-Regulated Lifestyle in Bacillus thuringiensis a11111 Thomas Dubois, Karoline Faegri, Se´bastien Ge´lis-Jeanvoine, Ste´phane Perchat, Christelle Lemy, Christophe Buisson, Christina Nielsen-LeRoux, Michel Gohar, Philippe Jacques, Nalini Ramarao, Leyla Slamti, Anne-Brit Kolstø, Didier Lereclus Through the analysis of the microarrays of the Bacillus thuringiensis (Bt) ΔkrsABC mutant strain, the authors have come to understand that the experiments presented in the original article on the role of the kurstakin resulted from a mistake in the mutant strain krsABC. An analysis of the krsABC mutant strain revealed that the disruption of the krsABC by a spc gene, conferring resistance to spectinomycin, was introduced in the Bacillus cereus strain ATCC14579 instead of the Bt strain 407 Cry-. The B. cereus strain ATCC14579 is closely related to the Bt strain 407 Cry- and is currently used in the authors’ laboratories for experi- ments focusing on B. cereus. A major difference between these two strains is the disruption of the nprR gene by a transposon in the B. cereus strain ATCC14579. As a result, this strain has naturally a NprR- phenotype and should therefore be unable to survive in the insect cadavers like the Bt mutant strain 407 ΔnprR ΔnprX [1]. Moreover, it was previously shown that the B. cereus strain ATCC14579 did not produce biofilm in LB peptone medium [2] and did not swarm on EPS agar, a low-nutrient medium [3]. The error in the experiments described in Figure 5B might therefore be due to the lack of a functional NprR regulator in the B. cereus strain ATCC14579 instead of the disruption of the krsABC genes in the Bt strain 407 Cry-. In order to check this hypothesis, the authors attempted to disrupt the krsABC genes in the Bt strain 407 Cry-. Several unsuccessful experi- ments suggested that it was impossible to disrupt these three genes by using their methodol- ogy. Thus, the authors decided to disrupt the krsC gene coding for a major synthetase of the NPRS system. The deletion of this gene abolishes the production of kurstakin [4]. To investi- gate the role of the kurstakin during the late stage of infection, the authors monitored the growth of the wild-type and ΔkrsC Bt 407 Cry- strains in insect larvae (Figure 1). The results showed that the deletion of the krsC gene and the subsequent lack of kurstakin did not affect the survival of the krsC mutant strain for at least 3 days in the insect cadaver. Supporting Information pp g S1 File. Marked-up PDF detailing changes. (PDF) S2 File. Clean Word version of revised article. (DOC) S3 File. Zip file containing renumbered Figures. (ZIP) S1 File. Marked-up PDF detailing changes. S1 File. Marked-up PDF detailing changes. (PDF) S1 File. Marked-up PDF detailing changes. (PDF) S2 File. Clean Word version of revised article. (DOC) S3 File. Zip file containing renumbered Figures. (ZIP) S3 File. Zip file containing renumbered Figures. (ZIP) 3. Constitutive expression of the krs operon involved in kurstakin production restored the survival of a nprR deficient mutant strain in the insect cadaver. 3. Constitutive expression of the krs operon involved in kurstakin production restored the survival of a nprR deficient mutant strain in the insect cadaver. 4. Necrotrophism is essential for the Bt infectious cycle, contributing to spore spreading. 4. Necrotrophism is essential for the Bt infectious cycle, contributing to spore spreading. To properly correct the original article, the authors have made substantial changes to the following sections: Author Summary, Results, Discussion, Materials and Methods, Figures, and References. Additional changes include updating of the affiliations of the authors listed on the original article to acknowledge where the work for this correction was performed. Ste´phane Perchat, Christelle Lemy, Christophe Buisson, Christina Nielsen-LeRoux, Michel Gohar, Nalini Ramarao, and Didier Lereclus are now affiliated with Micalis Institute, INRA, AgroParisTech, Universite´ Paris-Saclay, 78350 Jouy-en-Josas, France. The present address of Thomas Dubois is: Unite´ Mate´riaux et Transformations, CNRS, INRA, Universite´ de Lille, Villeneuve d’Ascq, France. His mail address is: Thomas.Dubois@lille.inra.fr. Further changes include the addition of Se´bastien Ge´lis-Jeanvoine and Leyla Slamti to the author list of this correction. Se´bastien Ge´lis-Jeanvoine is listed as the third author and his affiliation is Micalis Institute, INRA, AgroParisTech, Universite´ Paris-Saclay, 78350 Jouy-en- Josas, France. His mail address is: sebastien@gelis.ch. The contributions of this author are as follows: conceived and designed the experiments, performed the experiments and analyzed the data. SGJ declares no competing interests. SGJ received funding from the French “Ministère de l’Enseignement Supe´rieur et de la Recherche”. Leyla Slamti is listed as the eleventh author and her affiliation is Micalis Institute, INRA, AgroParisTech, Universite´ Paris-Saclay, 78350 Jouy-en-Josas. Her mail address is: Leyla.Slamti@jouy.inra.fr. The contributions of this author are as follows: conceived and designed the experiments, and analyzed the data. LS declares no competing interests. LS received funding from INRA. All changes to the original article are detailed in a marked-up PDF included as S1 File. A clean Word document version of the revised article is included as S2 File. The renumbered Figures, including the corrected Fig 5, are included as S3 File. PLOS Pathogens \| DOI:10.1371/journal.ppat.1006049 November 29, 2016 Published: November 29, 2016 2. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a lipopeptide named kurstakin. Copyright: © 2016 Dubois et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 3 PLOS Pathogens \| DOI:10.1371/journal.ppat.1006049 November 29, 2016 PLOS Pathogens \| DOI:10.1371/journal.ppat.1006049 November 29, 2016 3. Hsueh YH, Somers EB, Lereclus D, Ghelardi E, Wong AC. 2007. Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions. Appl Environ Microbiol. 73:7225–7231 doi: 10.1128/AEM.00690-07 PMID: 17921286 4. Ge´lis-Jeanvoine S, Canette A, Gohar M, Caradec T, Lemy C, et al. (2016) Genetic and functional analy- ses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis. Res Microbiol. doi: 10.1016/j.resmic.2016.06.002 PMID: 27353188 3. Hsueh YH, Somers EB, Lereclus D, Ghelardi E, Wong AC. 2007. Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions. Appl Environ Microbiol. 73:7225–7231 doi: 10.1128/AEM.00690-07 PMID: 17921286 4. Ge´lis-Jeanvoine S, Canette A, Gohar M, Caradec T, Lemy C, et al. (2016) Genetic and functional analy- ses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis. Res Microbiol. doi: 10.1016/j.resmic.2016.06.002 PMID: 27353188 References 1. Dubois T, Faegri K, Perchat S, Lemy C, Buisson C, Nielsen-LeRoux C, et al. (2012) Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis. PLoS Pathog 8(4): e1002629. doi: 10. 1371/journal.ppat.1002629 PMID: 22511867 2. Auger S, Krin E, Aymerich S, Gohar M. 2006. Autoinducer 2 affects biofilm formation by Bacillus cereus. Appl Environ Microbiol. 72:937–941 doi: 10.1128/AEM.72.1.937-941.2006 PMID: 16391139 1. Dubois T, Faegri K, Perchat S, Lemy C, Buisson C, Nielsen-LeRoux C, et al. (2012) Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis. PLoS Pathog 8(4): e1002629. doi: 10. 1371/journal.ppat.1002629 PMID: 22511867 1. Dubois T, Faegri K, Perchat S, Lemy C, Buisson C, Nielsen-LeRoux C, et al. (2012) Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis. PLoS Pathog 8(4): e1002629. doi: 10. 1371/journal.ppat.1002629 PMID: 22511867 2. Auger S, Krin E, Aymerich S, Gohar M. 2006. Autoinducer 2 affects biofilm formation by Bacillus cereus. Appl Environ Microbiol. 72:937–941 doi: 10.1128/AEM.72.1.937-941.2006 PMID: 16391139 2. Auger S, Krin E, Aymerich S, Gohar M. 2006. Autoinducer 2 affects biofilm formation by Bacillus cereus. Appl Environ Microbiol. 72:937–941 doi: 10.1128/AEM.72.1.937-941.2006 PMID: 16391139 2 / 3 PLOS Pathogens \| DOI:10.1371/journal.ppat.1006049 November 29, 2016 3 / 3
W2206185815.txt	https://www.scienceopen.com/document_file/c1bb362e-84fb-4440-8010-ed011f1e58c6/ScienceOpen/001_Pastore.pdf	en		Enabling Access to Astronomical Databases through the Grid: a Case Study	Electronic workshops in computing	2,004	cc-by	2,280	Enabling Access to Astronomical Databases through the Grid: a Case Study S. Pastore (1), A. Volpato (1), A. Baruffolo (1), L. Benacchio (1), G. Taffoni (2), R. Smareglia (2), C. Vuerli (2), R. Crevatin (2) (1) INAF – Osservatorio Astronomico di Padova, (2) INAF – Osservatorio Astronomico di Trieste Abstract In recent years great efforts have been spent in the astronomical community to allow a global and seamless electronic access to distributed astronomical data repositories, and to enable scientific analysis on them. A system capable of providing these services over distributed databases and computer resources is called a Virtual Observatory (VO). VOs are still in the study phase, with many projects around the world now delivering their first prototype implementations. In the meanwhile grid technologies have started to emerge and consolidate and are now expected to play a fundamental role in the development of Astrophysical Virtual Observatories. We report here about our activities aimed at integrating in the grid environment a system, developed in Padova, specifically designed for accessing very large astronomical catalogues. We evaluated several possible solutions, including the use of a tool for accessing databases developed by the European Data Grid (EDG) project, but finally we decided to adopt a web services based architecture, retaining the security infrastructure provided by the EDG software. We plan to evolve towards an implementation fully compliant with the latest Open Grid Service Architecture specification, but without loosing compatibility with the grid middleware adopted in our project. This work is being conducted in the framework of Grid.it, a project, funded by the Italian Ministry for Education, University and Research, aimed at studying a grid infrastructure for scientific research in Italy and developing specific software tools and protype applications that run on it. This catalogue access system will eventually become one of the first building blocks of the Italian Virtual Observatory, currently under development in the framework of a closely-related project, named DRACO. To this end, starting from the design phase of our system, and where applicable, we adopted the standards set forth by the International Virtual Observatory Alliance (IVOA). The framework The Astronomical Observatory of Padova (OAPd) Catalog group is involved in the Grid.it project, a multidisciplinary research project, funded by the Italian Ministry for Education, University and Research, aiming at “Enabling platforms for high-performance computational grids oriented to scalable virtual organizations”. The Grid.it project is organized around fifteen Work Packages (WPs) focused on implementing a grid infrastructure on a national scale (based upon the GARR scientific network), creating a grid-specific set of software tools and on developing some demonstrators within several applicative fields, ranging from Computational Chemistry to Earth Observation. WP10 (Grid Applications for Astrophysics) of this project collects the various groups whose main goal is exploring the use of grid technologies for the development of astrophysical applications. Within this work package the main goal of the OAPd group is to study the portability to the Grid of an existing system for the consultation of large astronomical catalogues, currently serving on the net the second Guide Start Catalog (GSC-II). In the same framework, OAPd closely collaborates with the Technology Group at the Astronomical Observatory of Trieste (OAT), which is dealing with the similar problem of integrating in the Grid the archive of observational data from the Italian Galileo National Telescope (TNG). The research field is constituted by Grid technologies: the set of hardware, protocols and software packages that enable coordination and sharing of highly distributed computational and storage resources (fig.1). Such environment supports, efficiently and in a secure and controlled way, resource sharing and integration in a scalable Virtual Organization (VO) context. VOs (sets of individuals or organizations that need to share resources to solve a given complex problem) have a strong dynamic structure, form, lasting and composition. The testbed for grid applications is the Italian INFN Production Grid for Scientific Applications (http://grid-it.cnaf.infn.it). Applications Programming High level Low level Networks Fig. 1: layers in the Grid.it infrastructure INFN-Grid middleware release 2.0.0 is derived from LCG (LHC Computing Grid) distribution, provided by the grid deployment project for High Physics applications related to the Large Hadron Collider. LCG itself is a stable version of the middleware released by EDG (European Data Grid) project. At present time, the production grid supports 18 sites and 13 Virtual Organization. Our Case Study Goal of the project is to integrate into the grid a system for accessing large astronomical catalogues. The existing system (fig.2) allows querying collections of catalogues by means of a web interface. The catalogue data and metadata are managed by an ORDBMS Object-relational database Management System (Informix Dynamic Server) enhanced by the PosAstro DataBlade, a module specifically developed for handling spatial information for astronomical objects that provides support for astronomical data types and an R-Tree based index to optimize query execution. The server side is implemented by a pool of java servlets; the use of CORBA technology allows interaction with legacy software. www clients Catalogues Catalogue Server Informix Database Server PosAstro Data Blade Gateways Internet Not-www clients Fig.2: existing GSC2 Access System Design-time considerations We decided to implement our prototype following the web services paradigm because: • this is one of the most stable and widely used architectures for building distributed applications; • the Grid reference architecture (Open Grid Services Architecture, OGSA) is based in turn on a web services extension; • although the Grid middleware of INFN production Grid doesn’t support services at present time, they will likely be included by the end of 2004; • currently it is under discussion within the Global Grid Forum (GGF) how OGSA will evolve in the near future, but since it will be most likely based on web services technology, our choice will not prevent us from following the evolution of architectural standards; • we want to be compliant with International Virtual Observatory Alliance (IVOA) standards: IVOA recommends the adoption of web services and XML for implementing data source access; • web services can be easily used as building blocks of more complex applications; • it is straightforward to develop user interfaces (e.g. by the means of web browser and JSP technology); • they can easily be integrated into existing Grid Portals. System architecture The system is composed by two web services: a Metadata Access service and a Query service (Fig. 3). The interaction between a client and the services is carried out by SOAP messages over https. Currently, the services can be accessed either interactively or in batch mode. Interactive access is performed through a JSP page that can be reached by a web browser. Batch access is carried out requesting the execution of a command line client application by submitting a Job Description Language (JDL) file from a machine with the User Interface Grid software installed. In both cases, the user must have a valid User Certificate installed on his machine. ?wsdl ?info ?wsdl SQL ADQL INFO SERVICE (InfoService) QUERY SERVICE (QueryService) Web Services Fig. 3 : current system architecture WSDL XML WSDL VOTable The services They are implemented in Java and both communicate via JDBC with the DBMS. • Metadata Access Service: retrieves metadata information about catalogues hosted by the system by connecting with a ‘lightweight’ database running on the same machine as the service container. This service answers to two possible requests: to the standard web services request ?wsdl with the WSDL file describing the service itself, and to the info request with an XML file containing the catalogue metadata in an IVOA compliant format. • Query Service: this service answers to queries expressed in SQL or ADQL with a VOtable containing the result set. Currently, for test purposes, queries are performed on a MySQL database containing a subset of the data hosted by the full system. It answers also to the standard web services request ?wsdl returning the WSDL file describing the service itself. Security Access to services is controlled following Grid Security Infrastructure (GSI) standard, based on the usage of X509 certificates both for Grid users and for Grid resources. We adopted the implementation provided by EDG Security: this package contains a modified version of Apache Tomcat (web application server) and Axis Engine (service container) along with the libraries needed to interact with User Certificates and the Grid middleware devoted to management of Virtual Organization membership. The EDG Securiry components are the Trust Manager and the Authorization Framework. Security = Authentication + Authorization The authentication of grid clients is performed by the Trust Manager on X.509 certificates basis. The Trust Manager is a pure Java-based component for validation of X.509 certificate paths used in SSL/TLS connections to secure web services. It is integrated at server side in the Tomcat servlet container, substitutes and extends the default SSLServerSocketFactory. The authorization is performed by the Authorization Framework, which makes the mapping from a grid client to an access role through the extraction of the subject Distinguished Name from the client certificate. The Authorization Framework consists of the Authorization Manager and a wrapper to integrate authorization functionality into the web services engine. In the request flow in front of the actual SOAP endpoint is put an AXIS handler that extracts the client certificate from the SOAP request, passes the subject DN and any optional interrogation parameters to the Authorization Manager, and stores the returned result in the MessageContext, which can then be retrieved by the protected service (Fig. 4). https://gridit.pd.astro.it/AstroServices.jsp SOAP request Authentication Trust Manager Grid Client grid-proxy-init + JDL job GSI-style (X.509) certificate Attribute (role) Axis Authorization Handler Authorization Manager Subject DN Security Policies Authorization Web services (QueryService, InfoService) XML output format Fig. 4 : interaction with EDG Security components Deployment Both services are installed on a computational node (Worker Node) connected to the Production Grid; they are deployed within Apache Tomcat web application server equipped with Axis, and secured by EDG security (Fig. 5). NETWORK ENVIRONMENT Catalogue Server [Web browser] with GSI-style certificate Web Services Service provider [Grid User] through UI Grid certificate SOAP Service requestor GSI Security WSDL [Secure grid/web services container] UDDI Service broker UDDI [Grid Information Service] GRID ENVIRONMENT Fig. 5. : system deployment and interaction with environment Work in progress In the current Grid architecture Information Services (discovery and monitoring of resources) are structured hierarchically. They encompass semantic and interaction models for computational (CE) and storage (SE) Grid resources. The corresponding models for data sources and services accessing them have not been defined yet. In close collaboration with CNAF the architecture of the Italian Production Grid is being extended in order to deal with this limitation. In this way it will be possible to publish and discover our data sources and services, achieving a true integration with the Grid infrastructure (Fig. 6). This process is being carried out in such a way that the description of data sources and services provided by the extended Grid Information Services will be allowed to be IVOA compliant whenever needed. The task of extending the Italian Grid middleware is being carried out at CNAF, while the Information Provider software for the newly defined grid resources is being developed at the Astronomical Observatory of Padova. LOCAL_Engine CATALOG AND SERVICE USER INTERFACE GENERIC USER_INTERFACE LDAP SERVER TOP_MSD_GIIS LOCAL_MSD_GRIS GRAM_CLIENT COMPUTING ELEMENT GRAM SERVER LOCAL RESOURCE_rdbms MANAGER WORKER NODE METADATA SOURCE ENGINE Fig. 6 : integration with the Grid Information Services Further developments The next step will exploit the true integration of the newly defined resources into the Grid to provide additional functionalities; it will then be possible for an application: • to save the output of a query on a Grid Storage Element either specified by the user or automatically chosen by the Resource Broker • to replicate the output file and to register the copies (notifying its presence to the Grid) in the most convenient locations for further processing Bibliography Albrecht, M, et al. 1996, "Astronomical Server URL'', http://vizier.u-strasbg.fr/doc/asu.html Baruffolo, A. & Benacchio, L. 1998a, in ASP Conf. Ser., Vol. 145, Astronomical Data Analysis Software and Systems VII, ed. R. 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https://openalex.org/W3164059597	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0251838&type=printable	English	null	Cross-cultural adaptation and psychometric properties of the Italian version of the Body Perception Questionnaire	PloS one	2,021	cc-by	8,220	PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Cerritelli F, Galli M, Consorti G, D’Alessandro G, Kolacz J, Porges SW (2021) Cross-cultural adaptation and psychometric properties of the Italian version of the Body Perception Questionnaire. PLoS ONE 16(5): e0251838. https://doi.org/10.1371/journal. pone.0251838 Background/Objective The purpose of this study was to cross-culturally adapt the Body Perception Questionnaire Short Form (BPQ-SF) into Italian and to assess its psychometric properties in a sample of Italian subjects. Editor: Antonio Palazo´n-Bru, Universidad Miguel Hernandez de Elche, SPAIN Received: December 30, 2019 Accepted: May 5, 2021 Published: May 27, 2021 Received: December 30, 2019 Accepted: May 5, 2021 Published: May 27, 2021 Cross-cultural adaptation and psychometric properties of the Italian version of the Body Perception Questionnaire Francesco CerritelliID1☯, Matteo Galli1,2☯, Giacomo ConsortiID1,3, Giandomenico D’AlessandroID1, Jacek Kolacz4,5, Stephen W. Porges5,6 1 Clinical-based Human Research Department, Foundation COME Collaboration, Pescara, Italy, 2 Research Department, SOMA, Istituto Osteopatia Milano, Milan, Italy, 3 Education Department of Osteopathy, Istituto Superiore di Osteopatia, Milan, Italy, 4 Socioneural Physiology Laboratory, Kinsey Institute, Indiana University, Bloomington, Indiana, United States of America, 5 Traumatic Stress Research Consortium, Kinsey Institute, Indiana University, Bloomington, Indiana, United States of America, 6 Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America ☯These authors contributed equally to this work. giacomo.consorti@gmail.com * giacomo.consorti@gmail.com Methods A forward-backward method was used for translation. 493 adults were recruited for psycho- metric analysis. Structural validity was assessed with confirmatory factor analysis and a hypothesis testing approach. Internal consistency was assessed by Cronbach’s alpha and McDonald’s omega. Measurement invariance analysis was applied with an age-matched American sample. 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.0251838 PLOS ONE PLOS ONE Introduction The central nervous system is continuously updating the status of bodily states and visceral organs. The subjective experience of the ongoing bottom-up flow from the body may be clus- tered into a construct called “body awareness” (i.e. body perception) [1]. Research in the field of functional neuroanatomy, neurophysiology, and psychiatry has progressively uncovered the physiological and neural processes related to the subjective body experiences related to body awareness [2–5]. Interoception, the sense of the physiological condition of the body, is a neural process through which information from organs and tissues is transmitted to the brain, form- ing a neural pathway through which body awareness emerges. Incoming afferent information informs the functional regulation of tissues and organs mainly through the activity of auto- nomic nervous system (ANS) [1]. Consistent with an evolutionary perspective, interoception and ANS activity are crucial for preserving body homeostasis. Interoceptive and ANS central neural networks have been maintained and elaborated over the course of the evolutionary encephalization process, reaching higher-order cortical areas (e.g., anterior insula) where sub- jective or mental body awareness emerges. Mental awareness of body homeostasis improves homeostatic preservation controlling emotional behaviour and social communication [6]. Body awareness, also called body perception, is defined as the ability to recognize internal body cues [7] including changes of target organs innervated by the ANS (i.e., autonomic reac- tivity) [1]. Body perception has been found to be useful in the management of chronic diseases such as chronic low back pain [8,9], congestive heart failure [10], chronic renal failure [11,12], and irritable bowel syndrome [13–16]. Body awareness is also important for physical stability and wellbeing [8,10–12]. Several research disciplines, including psychiatry and somatic-oriented therapies, have shown interest in the body perception construct. Specifically, there is an interest in how indices of subjective body experience can complement laboratory-based measures [16]. Moreover, in many clinical settings patient subjective reports (e.g., pain and other subjective symptoms) are important sources of information that patients and clinicians can use to evaluate therapy prog- ress and health status. Therefore, efforts have been made to create self-reported questionnaires to investigate body perception and autonomic reactivity, but few have shown strong psycho- metric properties with a theoretical coherence with the organization of peripheral neural path- ways [1]. PLOS ONE PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ Conclusions Funding: The authors received no specific funding for this work. Funding: The authors received no specific funding for this work. Our results support the Italian version of the BPQ as having consistent psychometric proper- ties in comparison with other languages. Competing interests: The authors have declared that no competing interests exist. Results Copyright: © 2021 Cerritelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The single-factor structure fit the awareness subscale (RMSEA = .036, CFI = .983, TLI = .982). Autonomic reactivity (ANSR) was well-described by supra- and sub-diaphragmatic subscales (RMSEA = .041, CFI = .984, TLI = .982). All subscales were positively correlated (r range: .50-.56) and had good internal consistency (McDonald’s Omega range: .86-.92, Cronbach’s alpha range: .88-.91). Measurement invariance analysis for the Awareness model showed significant results (p<0.001) in each step (weak, strong and strict) whereas the ANSR showed significant results (p<0.001) only for the strong and strict steps. Data Availability Statement: All relevant data are within the paper and its Supporting information files. 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 Participants Four hundred and ninety three adults were recruited to complete the questionnaire during clinical visits with professional osteopaths. Therefore, the current sample might not be consid- ered a regular community sample, but rather an osteopathic care sample. A comprehensive description of participants’ characteristics is reported in Table 1. The target sample size was calculated taking into account at least 10 participants for each item of the questionnaire [20]. Data collection forms were designed at the Foundation COME Collaboration international coordinating center in Pescara, Italy. Participants completed the approximately 10-minute questionnaire online through the Google Forms platform, in the presence of the osteopath in their private practice. There was no financial incentive for completion. Subjects reported socio-demographic and clinical characteristics, gender, age, height, weight, education level, work, annual income, medication usage, physical activity, smoking habits, pathologies. Questions concerning the medication use, physical activity and smoking habits allowed only dichotomous answers (yes/no) without quantitative specification, apart from the physical activity question (if 2/week). They also completed the Italian translation of the BPQ-SF (BPQ-I). Introduction The Body Perception Questionnaire (BPQ) [17] is a self-reported questionnaire developed to assess the subjective experiences of the function and reactivity of target organs and struc- tures that are innervated by the ANS. Its development has followed the theoretical division of the ANS described by the polyvagal theory [18]. The polyvagal theory is an evolutionary neurophysiological framework that divides the vagal circuits within the parasympathetic ner- vous system into a ventral vagal complex (VVC) and a dorsal vagal complex (DVC). VVC reg- ulates the striated muscles of the face, head, and visceral organs above the diaphragm through efferent nerves that originate from the nucleus ambiguous in the brainstem; the physiological status of the VVC targeted organs is represented through sensory pathways that terminate in the nucleus of the solitary tract (NTS) in the brainstem. DVC efferent neurons originating in PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 2 / 15 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ the dorsal nucleus of the vagus regulate the organs below the diaphragm while the physiologi- cal status of these organs is represented in NTS through afferent vagal fibers. The original BPQ was composed of 122 items, assessing body awareness, autonomic ner- vous system reactivity, cognitive-emotional-somatic stress response, body and cognitive stress response styles, and health history. Since its introduction, the BPQ has been frequently used in clinical research and translated into several languages [1,19]. However, recently a shorter ver- sion of the questionnaire was developed and validated, focusing primarily on two subscales: (1) Awareness (26 items); (2) Autonomic Nervous System Reactivity (ANSR) (20 items) [1]. The purpose of this study was to cross culturally adapt the BPQ into Italian to assess its The purpose of this study was to cross-culturally adapt the BPQ into Italian, to assess its psychometrics characteristics in a sample of Italian subjects, and to examine associations between the subscales of BPQ and the sample characteristics. Materials and methods This psychometric study consisted of a cross-cultural adaptation and factor analysis of the Body Perception Questionnaire (BPQ). The study protocol was approved by the Internal Review Board of the Foundation Centre for Osteopathic Medicine Collaboration (COME IRB n.01/2019). All subjects gave their written consent and all procedures followed the Declaration of Helsinki. The Body Perception Questionnaire (BPQ) The Body Perception Questionnaire Short Form (BPQ-SF) is a self-report measure of the body awareness and experiences of autonomic reactivity [1,17]. It has demonstrated strong psycho- metric properties and a consistent factor structure across multiple languages [1,19]. It is com- posed of two domains: body awareness (26 items) and autonomic nervous system reactivity (ANSR; 20 items). The body awareness domain measures sensitivity to internal bodily func- tions (e.g. “During most situations I am aware of my mouth being dry.”). The Autonomic Ner- vous System Reactivity (ANSR) domain is composed of a supradiaphragmatic reactivity subscale, which measures the typical experience of body reactions above the diaphragm (e.g. “During most situations I am aware of sweat in my armpits”), and a subdiaphragmatic reactiv- ity subscale, which measures of gastrointestinal functions below the diaphragm (e.g. “During most situations I am constipated”). There is an item—“I feel like vomiting”—that is included 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ Table 1. Sample characteristics. Characteristics Values Gender (%) F: 292 (59.23) M: 201 (40.77) Age (SD) 34.71 (14) BMI (SD) 23.46 (3.86) Education level (%) High school diploma: 247 (50.10) University degree: 200 (40.57) Other: 46 (9.33) Smoker (%) yes: 126 (25.56) no: 367 (74.44) Medications usage (%) yes: 119 (24.14) no: 374 (75.86) Psychiatric disorder (%) yes: 4 (0.81) no: 489 (99.19) Physical activity 2/week (%) yes: 289 (58.62) no: 204 (41.38) Other Diseases (%) yes: 146 (29.61) no: 347 (70.39) https://doi.org/10.1371/journal.pone.0251838.t001 https://doi.org/10.1371/journal.pone.0251838.t001 in both the supradiaphragmatic reactivity and the subdiaphragmatic reactivity domains. Responses measure frequency of sensations, assessed on a 5-point Likert-type scale (“Never” to “Always”). in both the supradiaphragmatic reactivity and the subdiaphragmatic reactivity domains. in both the supradiaphragmatic reactivity and the subdiaphragmatic reactivity domains. Responses measure frequency of sensations, assessed on a 5-point Likert-type scale (“Never” to “Always”). p p g y p g y Responses measure frequency of sensations, assessed on a 5-point Likert-type scale (“Never” to “Always”). Data preparation Confirmatory Factor Analysis (CFA) was performed on dichotomized items (0 = never, 1 = occasionally or more often) to maintain acceptable response cell sizes and replicate meth- ods used with the BPQ in other languages [21]. Psychometric assessment The Consensus-based Standards for the selection of health Measurement Instruments (COS- MIN) initiative [22] and the International Society for Quality of Life Research (ISOQOL) [23] were used as methodological guidelines. Validity refers to the degree to which a patient- reported instrument measures the construct(s) it purports to measure [23]. Two subdomains of validity were assessed in this study: structural validity, referred as the degree to which the scores of an instrument are an adequate reflection of the dimensionality of the construct to be measured, and construct validity/hypotheses testing, that is the degree to which the scores of an Health Related Patient-Reported Outcome instrument (HR-PRO) are consistent with hypotheses (for instance with regard to internal relationships, relationships to scores of other instruments, or differences between relevant groups) based on the assumption that the instru- ment validly measures the construct to be measured [1]. Exploratory factor analysis was com- puted on full-score items to assess the number of adequate factors, which was too elevated in previous studies and constrained Cabrera et al. to adopt dichotomization. Factor structure was assessed for convergence with previously reported dimensionality in English- and Spanish-lan- guage samples [1] using confirmatory factor analysis. The hypotheses were that the age was normally distributed with respect to the Awareness subscale values and that age was negatively correlated with ANSR subscale value [24,25]. The hypothesis regarding physical activity was that it was positively correlated with Awareness and ANSR subscales values [26,27]. Furthermore, we hypothesized that Smoking habits [28], BMI [25], and educational level [29] are associated with the Awareness Subscale values. Cross-cultural adaptation The cross-cultural adaptation followed a previously used method for the adaptation of the same questionnaire in another language [1]. First, two native Italian speakers fluent in English independently translated the BPQ. One translator was an Italian professional with a medical background fluent in English. The other was a professional English-Italian translator with 20-year translation experience and no medical background. A common forward translated version was agreed upon by the two translators. Second, the provisional BPQ Italian version was independently back-translated by two English native speakers who were fluent in Italian. Third, all the translations and the provisional BPQ were discussed by an expert committee including the translators, a linguistic expert, two osteopaths, and one epidemiologist. The committee discussed the content by comparing semantic, idiomatic, experiential, and concep- tual equivalence; the goal was to develop the pre-final Italian BPQ (BPQ-I) to be understand- able to a reading level of a typical 18 years old. Fourth, the BPQ-I was pilot-tested with 20 healthy subjects (62% female; mean age 36.4 ± 6.6 years). After BPQ-I completion, subjects were asked to report any items with unclear meaning. All reported comprehension problems were discussed by three authors (FC, GC, GdA) and used to inform modifications of the BPQ-I. 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ Statistics Data were analyzed using different measures in relation to the type of data (continuous, ordi- nal, categorical and dichotomous). Mean, median, mode, point estimates, range, standard deviation and 95% confidence intervals were used. Data analysis was conducted using R ver- sion 3.5.1 (R Core Team 2017) and Rstudio Version 1.1.463 (RStudio, Inc 2009–2018). Hypothesis tests were conducted using a critical alpha value of 0.05. PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 Factor Analysis and Measurement Invariance Analysis (MIA) Confirmatory factor analysis was conducted using the R package “lavaan” [30]. Goodness of fit to the data was evaluated using root mean squared error of approximation (RMSEA), the Tucker-Lewis Index (TLI), and the Comparative Fit Index (CFI) [31–34]. We interpreted good fit to be evidenced by an RMSEA value near .06 or lower as well as CFI and TLI values near .95 or greater, as recommended by Hu and Bentler [35]. We used variance-covariance matrices, which dimensions corresponded the same as the items included per each model (Awareness model = 26x26; ANSR model = 20x20). Weighted Least Square Mean and Variance (WLSMV) was used as estimator according to Barendse et al. [36] that suggested using models with dis- crete responses [37]. Correlations between factors were not constrained, an analysis decision that can reproduce correlated or uncorrelated factor structures. Based on results from prior factor analysis on BPQ in other languages, CFA was performed to examine the fit of the (1) one-factor solution for the Body Awareness domain; (2) a two-factor solution for the PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 5 / 15 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ Autonomic Reactivity domain with supra-diaphragmatic and sub-diaphragmatic domains, uncorrelated each other. Based on the findings of Cabrera et al. [1] the item “I feel like vomit- ing” was included in both ASNR Supra and Sub-diaphragmatic subscale. Furthermore, we used an age-matched American sample to assess measurement invariance of the factor structure between Italian and American responses. The American sample was recruited online and its collection and descriptive statistics are described in Cabrera et al. [1] study. To conduct the analysis, we used the measurement Invariance function of “semTools” R package [38], which performs multiple group analyses with increasing restrictions on parame- ters, from configural to strict invariance using the Chi-squared difference test, RMSEA, and CFI. Statistical significance of the Chi-squared difference test indicates that exact fit of the model has to be rejected. However, when sample size is large, small differences between observed and model-implied parameters can result in rejection of the model. To avoid hypoth- esis testing over-sensitivity due to high power, we also used the RMSEA, where values smaller than .05 indicate close fit, and values smaller than .08 are considered satisfactory. CFI values over .95 indicate also a reasonably good fit. Internal consistency Internal consistency is defined as the degree to which the items measure the same construct, based on their interrelatedness [22]. The Cronbach’s alpha [40] and McDonald’s omega were calculated using the R “psych” package [41]. McDonald’s omega was selected because it showed to be a stronger index in case of categorical items and variable factor loadings as with BPQ-I items [42]. Internal consistency was computed for each subscale separately. Association of BPQ with demographic and clinical variables Subscale scores were calculated and examined for association of demographic and clinical vari- ables. Associations of BPQ scores with age and BMI were calculated using Kendall and Pear- son’s correlation coefficients. Categorical demographic variables were compared using Welch two sample t-tests. In linear regressions we set each one with the respective subscales of the BPQ (Awareness, ANSR Supra and Sub-diaphragmatic scores) as the dependent variables and the demographic and clinical characteristics of the sample as independent variables (age, gender, physical activ- ity and medication use), selecting the appropriate model using the stepwise methods. ANOVA was used to evaluate associations of the BPQ subscales with education. Factor Analysis and Measurement Invariance Analysis (MIA) CFI changes of .02 and RMSEA of .03 are most appropriate for tests of weak invariance with large group sizes, and variations of -.01 for ΔCFI and .01 for ΔRMSEA are appropriate for strong invariance tests. Partial invariance is evi- denced if the majority of items on the factor are invariant [39]. Floor and ceiling effects Floor and ceiling effects were considered to be present if 15% of the patients reported the lowest (0) or highest (46 possible BPQ score. The effect was considered also for the BPQ sub- scales separately [43]. Internal consistency Results of McDonald’s Omega were calculated for the Body Awareness subscale (0.92; CI: 0.90, 0.95), Supradiaphragmatic Reactivity (0.88; CI: 0.84, 0.90) and Subdiaphragmatic Reactivity (0.86; CI: 0.83, 0.89). Cronbach’s Alpha results were also calculated for the Body Awareness subscale (0.91; CI: 0.90, 0.92), Supradiaphragmatic Reactivity (0.88; CI: 0.86, 0.90) and Sub- diaphragmatic Reactivity (0.78; CI: 0.75, 0.81). Descriptive statistics The BPQ-SF was scored by adding the dichotomized responses (0 = never, 1 = occasionally or more often) in accordance to the factor structure described above. Descriptive statistics for the resulting scores are in Table 4. The sample mean score for the Awareness subscale was 19.3 (±6.05), for the supradiaphragmatic subscale of the ANSR was 6.3 (±4.41) and for the sub- diaphragmatic subscale of the ANSR was 3.71 (±1.96). Confirmatory Factor Analysis (CFA) and Measurement Invariance Analysis (MIA) Confirmatory factor analysis showed that the single-factor structure fit the awareness subscale well (RMSEA = 0.036, CFI = 0.983, TLI = 0.982). Autonomic reactivity was well-described by 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ supra- and sub-diaphragmatic subscales (RMSEA = 0.041, CFI = 0.984, TLI = 0.982; Table 2). Supra- and sub-diaphragmatic reactivity were positively correlated (r = 0.56) and were respec- tively related to Awareness (r = 0.56, r = 0.50). All these correlations showed statistical signifi- cance (p< 0.001). To examine the possibility of other potential well-fitting factor structures, a post hoc exploratory factor analysis was performed on the raw (non-dichotomized items). The results did not reveal any novel well-fitting factor structure beyond that tested by the CFA (see supporting information). Measurement invariance analysis showed that Chi-Squared difference test for Awareness model results significant in each step (weak, strong and strict) with p-values <0.001. Consider- ing the RMSEA, values were 0.075 for the configural and weak invariance and 0.085 for the strong and strict ones. The CFI values were 0.85 for the configural and 0.84 for the weak invariance, 0.79 for the strong and 0.78 for the strict ones. Analyzing the ANSR model, the “weak” step of Measurement Invariance was not significant (p = 0.55), whereas all the other steps (strong and strict invariances) showed significant values (p-value < 0.001). The RMSEA models produced values of 0.067 for the configural, 0.065 for the weak invariance, 0.069 for the strong and 0.071 for the strict one. The CFI values were 0.90 for the configural and for the weak invariance, .88 for the strong and for the strict ones. Table 3 reports results from Measurement Invariance Analysis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE PLOS ONE Table 2. Confirmatory Factor Analysis (CFA) response per items. Associations with demographic variables Correlations between BPQ-SF subscales with Age and BMI were calculated using Pearson and Kendall correlation coefficients (Table 5). Results showed negative correlations between age and BPQ-SF awareness and sub-diaphragmatic reactivity. A linear regression model showed that males had lower awareness values compared to females (β = -1.28, CI: -2.39, -0.31; p = 0.02) and there was a significant negative association with age (β = -0.06, CI: -0.09, -0.02; p = 0.002; S2 Table in S1 File). ANSR supradiaphragmatic reactivity showed a negative association with age (β = -0.03, CI: -0.06, -0.004; p = 0.02), lower scores in males (β = -1.05, CI: -1.81, -0.29; p = 0.007) and lower scores for those who were physical active (β = -1.66, CI:-2.43, -0.89; p<0.001). A positive association was found with medication use (β = 0.97, CI:0.12, 1.82; p = 0.03; S3 Table in S1 File). The ANSR subdiaphrag- matic reactivity linear regression model showed a negative association with age (β = -0.02, CI: -0.03, -0.005; p = 0.004), lower scores among males (β = -0.6, CI: -0.93, -0.26; p = 0.0005) and lower scores in those who were physical active (β = -0.44, CI: -0.79, -0.1; p = 0.01; S4 Table in S1 File). 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ PLOS ONE Table 2. (Continued) BPQ Items Estimate Standard Error z Value P “I have indigestion” 2.400 0.456 5.265 0.000 “After eating I have digestive problems” 2.094 0.407 5.148 0.000 “I have diarrhea” 1.880 0.377 4.985 0.000 https://doi.org/10.1371/journal.pone.0251838.t002 Table 3. Measurement invariance analysis per awareness and ANSR model. Steps Awareness Model ANSR Model CFI RMSEA P(>\|χ2\|) CFI RMSEA P(>\|χ2\|) Configural 0.85 0.075 - 0.90 0.067 - Weak 0.84 0.075 0.0001 0.90 0.065 0.55 Strong 0.79 0.085 0.0001 0.88 0.069 0.0001 Strict 0.78 0.085 0.0001 0.88 0.071 0.0001 Table shows the results of the Measurement Invariance Analysis for Awareness and ANSR models. The rows are the steps of testing invariance in order to increase constrains (from Configural to Strict). The columns show the values of Comparative Fit Index (CFI), Root Mean Squared Error of Approximation (RMSEA) and of P- value related to Chi square tests (P(>\|χ2\|)). Table 2. (Continued) BPQ Items Estimate Standard Error z Value P “I have indigestion” 2.400 0.456 5.265 0.000 “After eating I have digestive problems” 2.094 0.407 5.148 0.000 “I have diarrhea” 1.880 0.377 4.985 0.000 https://doi.org/10.1371/journal.pone.0251838.t002 Table 3. Measurement invariance analysis per awareness and ANSR model. Steps Awareness Model ANSR Model CFI RMSEA P(>\|χ2\|) CFI RMSEA P(>\|χ2\|) Configural 0.85 0.075 - 0.90 0.067 - Weak 0.84 0.075 0.0001 0.90 0.065 0.55 Strong 0.79 0.085 0.0001 0.88 0.069 0.0001 Strict 0.78 0.085 0.0001 0.88 0.071 0.0001 Table shows the results of the Measurement Invariance Analysis for Awareness and ANSR models. The rows are the steps of testing invariance in order to increase constrains (from Configural to Strict). The columns show the values of Comparative Fit Index (CFI), Root Mean Squared Error of Approximation (RMSEA) and of P- value related to Chi square tests (P(>\|χ2\|)). Table 3. Measurement invariance analysis per awareness and ANSR model. Steps Awareness Model ANSR Model CFI RMSEA P(>\|χ2\|) CFI RMSEA P(>\|χ2\|) Configural 0.85 0.075 - 0.90 0.067 - Weak 0.84 0.075 0.0001 0.90 0.065 0.55 Strong 0.79 0.085 0.0001 0.88 0.069 0.0001 Strict 0.78 0.085 0.0001 0.88 0.071 0.0001 Table 3. Measurement invariance analysis per awareness and ANSR model. Table shows the results of the Measurement Invariance Analysis for Awareness and ANSR models. The rows are the steps of testing invariance in order to increase constrains (from Configural to Strict). PLOS ONE The columns show the values of Comparative Fit Index (CFI), Root Mean Squared Error of Approximation (RMSEA) and of P- value related to Chi square tests (P(>\|χ2\|)). PLOS ONE BPQ Items Estimate Standard Error z Value P Awareness “Swallowing frequently” 1.000 “An urge to cough to clear my throat” 1.026 0.110 9.345 0.000 “My mouth being dry” 1.107 0.125 8.829 0.000 “How fast I am breathing” 1.196 0.125 9.589 0.000 “Watering or tearing of my eyes” 1.190 0.122 9.763 0.000 “Noises associated with my digestion” 0.820 0.100 8.211 0.000 “A swelling of my body or parts of my body” 0.814 0.113 7.218 0.000 “An urge to defecate” 0.933 0.121 7.724 0.000 “Muscle tension in my arms and legs” 0.988 0.127 7.806 0.000 “A bloated feeling because of water retention” 1.032 0.129 8.006 0.000 “Muscle tension in my face” 1.400 0.146 9.584 0.000 “Goose bumps” 1.116 0.120 9.331 0.000 “Stomach and gut pains” 0.894 0.129 6.934 0.000 “Stomach distension or bloatedness” 1.011 0.127 7.988 0.000 “Palms sweating” 1.466 0.167 8.791 0.000 “Sweat on my forehead” 1.538 0.164 9.359 0.000 “Tremor in my lips” 1.655 0.183 9.048 0.000 “Sweat in my armpits” 0.947 0.113 8.374 0.000 “The temperature of my face (especially my ears)” 1.247 0.143 8.706 0.000 “Grinding my teeth” 1.182 0.152 7.771 0.000 “General jitteriness” 0.766 0.108 7.101 0.000 “The hair on the back of my neck standing up” 1.293 0.146 8.838 0.000 “Difficulty in focusing” 0.736 0.104 7.066 0.000 “An urge to swallow” 1.515 0.141 10.736 0.000 “How hard my heart is beating” 1.095 0.131 8.384 0.000 “Feeling constipated” 1.139 0134 8.488 0.000 ANSR Supradiaphragmatic “I have difficulty coordinating breathing and eating” 1.000 “When I am eating, I have difficulty talking” 0.938 0.074 12.602 0.000 “My heart often beats irregularly” 0.909 0.080 11.371 0.000 “When I eat, food feels dry and sticks to my mouth and throat” 0.995 0.073 13.692 0.000 “I feel shortness of breath” 0.975 0.081 12.095 0.000 “I have difficulty coordinating breathing with talking” 1.049 0.068 15.435 0.000 “When I eat, I have difficulty coordinating swallowing, chewing, and/or sucking with breathing” 1.012 0.063 16.160 0.000 “I have a persistent cough that interferes with my talking and eating” 0.879 0.072 12.131 0.000 “I gag from the saliva in my mouth” 1.061 0.071 15.012 0.000 “I have chest pains” 0.966 0.076 12.753 0.000 “I gag when I eat” 0.997 0.072 13.807 0.000 “When I talk, I often feel I should cough or swallow the saliva in my mouth” 1.071 0.080 13.336 0.000 “When I breathe, I feel like I cannot get enough oxygen” 1.077 0.077 13.908 0.000 “I have difficulty controlling my eyes” 0.848 0.077 11.017 0.000 “I feel like vomiting” 0.684 0.092 7.466 0.000 ASNR Subdiaphragmatic “I feel like vomiting” 1.000 0.000 “I have ’sour’ stomach” 1.963 0.392 5.013 0.000 “I am constipated” 2 097 0 407 5 151 0 000 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 8 / 15 Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ Discussion The aims of the present study were to adapt the BPQ into Italian language, to assess its psycho- metric characteristics in a sample of Italian subjects, and examine the associations between the subscales of BPQ and the sample characteristics. The confirmatory factor analysis showed comparable results to the English, Spanish, and Chinese versions previously described in the literature [1,19], with subscales reflecting body awareness, supradiaphragmatic autonomic reactivity, and subdiaphragmatic autonomic reactivity. All subscales also demonstrated strong internal consistency. A post hoc exploratory factor analysis did not suggest an alternative factor structure differ- ent from that tested in this confirmatory factor analysis and previous studies. In the initial BPQ psychometric study [1], factor analysis on the full item distributions (5 ordered categories using polychoric correlations) required the estimation of an excess number of parameters with initial results suggesting that the full response item loadings were resulting in overfitting of the data and large influence of random noise. The solution employed by those authors was to cre- ate binary cut offs that would be less sensitive to noise and overfitting the data, which pro- duced reliable, interpretable factor solutions that could be replicated across samples. However, this left unresolved questions about whether the factor structure could be replicated with the full item distributions. In this study, the post-hoc EFA conducted on the full-item distributions did not reveal a better fitting factor structure and thus supported the factor structure that has been previously described. The results of the current analysis demonstrated partial measurement invariance between the two BPQ subscales of the English and the Italian version. In order to understand the possi- ble reasons for this partial invariance, methodological and cultural factors need to be consid- ered. It has been argued that MIA is influenced by the sample and model size. Putnick and Bornstein [39] affirmed that in large samples (N> 100) the chi-squared test increases its power to reject the null hypothesis. In the case of the present study, therefore, the total sample was 1009 (Italian = 493, U.S.A. = 516), supporting the assertion that statistical power was very high and capable of identifying small, possibly insubstantial differences. Another important factor that influences the MIA is model size. According to Putnick and Bornstein [39], smaller mod- els (e.g. composed by 4 factors with 2 indicators) are more likely to show more sensitive CFI and RMSEA indices than larger models (e.g. Floor and ceiling effects Extreme floor or ceiling effects were not observed in our sample according to the 15% crite- rion. The lowest score (0) was presented in 2 subjects of our sample (0.41%), whereas the high- est score (46) was reported in 26 subjects (5.27%). Table 4. Descriptive statistics of BPQ-SF score. Mean Median SD Skew Kurtosis Min Max BPQ-SF Body Awareness 19.3 21.0 6.05 -0.90 0.30 0.0 26.0 BPQ-SF Supradiaphragmatic Reactivity 6.3 6.0 4.41 0.42 -0.82 0.0 15.0 BPQ-SF Subdiaphragmatic Reactivity 3.7 4.0 1.96 -0.497 -0.98 0.0 6.0 https://doi.org/10.1371/journal.pone.0251838.t004 Table 5. Pearson and Kendall’s correlation indices. Pearson Kendall Age and BPQ-SF Awareness -0.0113 -0.079 Age and BPQ-SF ANSR Supra: -0.037 Supra: -0.078() Sub: -0.10() Sub: -0.09() BMI and Awareness 0.014 -0.004 BMI and ANSR Supra: -0.002 Supra: -0.029 Sub: -0.025 Sub: -0.062 () p-value <0.05; () p-value<0.01; () p-value<0.001. Finally, an ANOVA model did not show association between education levels and the Awareness subscale score (F = 1.48; p = 0.22). https://doi.org/10.1371/journal.pone.0251838.t005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 9 / 15 Table 4. Descriptive statistics of BPQ-SF score. Mean Median SD Skew Kurtosis Min Max BPQ-SF Body Awareness 19.3 21.0 6.05 -0.90 0.30 0.0 26.0 BPQ-SF Supradiaphragmatic Reactivity 6.3 6.0 4.41 0.42 -0.82 0.0 15.0 BPQ-SF Subdiaphragmatic Reactivity 3.7 4.0 1.96 -0.497 -0.98 0.0 6.0 https://doi.org/10.1371/journal.pone.0251838.t004 Table 4. Descriptive statistics of BPQ-SF score. Table 4. Descriptive statistics of BPQ-SF score. Table 5. Pearson and Kendall’s correlation indices. Pearson Kendall Age and BPQ-SF Awareness -0.0113 -0.079 Age and BPQ-SF ANSR Supra: -0.037 Supra: -0.078() Sub: -0.10() Sub: -0.09() BMI and Awareness 0.014 -0.004 BMI and ANSR Supra: -0.002 Supra: -0.029 Sub: -0.025 Sub: -0.062 () p-value <0.05; () p-value<0.01; () p-value<0.001. Finally, an ANOVA model did not show association between education levels and the Awareness subscale score (F = 1.48; p = 0.22). https://doi.org/10.1371/journal.pone.0251838.t005 Table 5. Pearson and Kendall’s correlation indices. 9 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 Discussion 4 factors with 6 indicators or larger). Considering the model size of the current research (Awareness = 26 items; ANSR = 20 items), it is evident that the MIA is characterized by a large model. As a consequence, the CFI and RMSEA indices might be influenced by this methodological element and therefore the results need to be con- sidered in relation to the model size. It is worth noting that comparing two samples from different countries might lead to addi- tional bias. Indeed, another factor to explain the partial invariance should be sought in the cul- tural differences present in the two populations. Differences possibly present also in the reading and understanding of the BPQ-SF items, despite the fact that the translation from English to Italian followed the WHO international guidelines [44]. For these reasons, we argue that the results of the MIA likely partially reflect the cross-cultural adaptation of the Italian ver- sion of the BPQ-SF. Moreover, since the study did not include convergent or discriminant validity testing the study can only be used to inform understanding of the dimensionality of the Italian BPQ-SF. Negative associations were found between ANSR subscale and age, physical activity, and male gender. Similarly, a negative association between age and male gender and the awareness subscale was observed. Positive associations were found between medication use and ANSR supradiaphragmatic subscale (S3 Table in S1 File). Our findings are in accord with Cabrera 10 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ et al. [1] and with several previous works [24,45] which found a decrease of interoception and cardiac autonomic regulation by the ventral vagal complex [25,46,47] in elderly people. Physi- cal activity seems to have no influence on the Awareness subscale, which is not consistent with other studies using different instruments of body awareness [26,27]. This difference might depend on the instrument used for the measurement. In fact, Multidimensional Assessment of Interoceptive Awareness is a questionnaire that measures interoception using a distinct frame- work from the BPQ. Some authors describe interoception as encompassing three distinct dimensions (accuracy, sensibility, and awareness) [48,49]. According to these authors, the BPQ Body Awareness subscale measures interoceptive sensibility. The different facets of the same construct measured could justify the observed difference between our result and other studies [26,49]. Discussion Since some of our findings differ from the prior literature relating to physical activity, further investigations are needed to examine relations between physical activity and Awareness and ANSR. However, one potential hypothesis to explain the absence of link between physical activity and body awareness might be based on reflecting a ‘body numbness’, that is a feeling of harmless sensation, which has been shown to be associated with those who exercise as their primary mode to regulate bodily state [29]. Smoking was not associated with differences in any BPQ subscales. Our results are not con- sistent with the hypothesis proposed by Naqvi and Bechara [28]: the interpretation framework between the awareness of the interoception and smoking takes into account that the insula— which can be affected by nicotine making the interoceptive information available to conscious awareness. Since the role of the insula in processes related to conscious interoception is estab- lished [50,51], the observed correlation did not converge with prior evidence and theory. In general, our results showed a significant difference on ANSR supradiaphragmatic score between participants who are using medication compared to participants who are not. This finding is in line with previous studies [52] showing interoceptive alteration following medica- tion usage. All the subscale scores were higher in women, compared to men. This finding is consistent with Cabrera et al. [1] suggesting robustness to cultural and language differences of the BPQ [46,53]. The results of the Welch two sample t-test between gender showed a signifi- cant difference among Male and Female groups in all BPQ subscales, consistent with those of Antelmi et al. study [46]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 Project administration: Francesco Cerritelli. Resources: Francesco Cerritelli. Supervision: Francesco Cerritelli, Jacek Kolacz, Stephen W. Porges. Writing – original draft: Francesco Cerritelli, Matteo Galli, Giacomo Consorti, Giandome- nico D’Alessandro. Writing – review & editing: Francesco Cerritelli, Matteo Galli, Giacomo Consorti, Giandome- nico D’Alessandro, Jacek Kolacz, Stephen W. Porges. Limitations and recommendations for future studies The present study includes some limitations. The psychometric evaluation of the questionnaire did not encompass the assessment of convergent and divergent validity. Therefore, it is not possible to affirm that the BPQ-I actually measures the intended constructs (i.e. body aware- ness and autonomic reactivity). Although the body awareness construct has been studied in previous studies [49], the autonomic reactivity construct still needs to be tested with sensor- based measures. Further studies are needed to better define those constructs and their proper- ties. Participants were recruited from a pool of clients seeking osteopathic care. Therefore, the current sample is not a regular community sample, but rather an osteopathic care sample, where a significant percentage of subjects reported a health-related condition. It is possible that participants in this sample may have higher autonomic reactivity or lower awareness as part of their reason for seeking care. Further studies need to be conducted with more general samples to establish better normative data and replicate the psychometric features of the scale. Furthermore, the participants in the sample were highly educated (40% with a university degree). A previous study underlined how education level might impact the body awareness of subjects with a direct correlation [54]. However, the sample in the study was specific and PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 11 / 15 PLOS ONE Cross-cultural adaptation and psychometric properties of the Italian version of the BPQ therefore the results might be difficult to generalize. In order to understand the relationship between education level and body awareness further studies are needed. Conclusions Our results support the Italian version of the BPQ as having consistent psychometric proper- ties in comparison with other languages. Applying the BPQ-I might help to identify the status of individual circuits that contribute to dysfunction and the development of novel interven- tions that can target autonomic or interoceptive dysfunction [1]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0251838 May 27, 2021 Author Contributions Conceptualization: Francesco Cerritelli, Giacomo Consorti, Giandomenico D’Alessandro, Jacek Kolacz, Stephen W. Porges. Conceptualization: Francesco Cerritelli, Giacomo Consorti, Giandomenico D’Alessandro, Jacek Kolacz, Stephen W. Porges. Data curation: Francesco Cerritelli, Matteo Galli, Jacek Kolacz, Stephen W. Porges. Formal analysis: Francesco Cerritelli, Matteo Galli, Giacomo Consorti. Investigation: Francesco Cerritelli, Giacomo Consorti, Giandomenico D’Alessandro. Methodology: Francesco Cerritelli, Giacomo Consorti, Giandomenico D’Alessandro, Jacek Kolacz, Stephen W. Porges. Project administration: Francesco Cerritelli. Project administration: Francesco Cerritelli. References 1. Cabrera A, Kolacz J, Pailhez G, Bulbena-Cabre A, Bulbena A, Porges SW. 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W4393145219.txt	https://elibrary.vdi-verlag.de/10.37544/1436-4980-2024-03-38.pdf	de		Methodik zum effizienten Einsatz von Sprachsteuerung/Methodology for the efficient use of voice control	Werkstattstechnik	2,024	cc-by	3,029	T I T E LT H E M A – FACHAUFSATZ Bei diesem Beitrag handelt es sich um einen wissenschaftlich begutachteten und freigegebenen Fachaufsatz („reviewed paper“). doi.org/10.37544/1436–4980–2024–03–38 Industrie 5.0: Sprachsteuerung als nutzerzentriertes Human-Machine-Interface (HMI) Methodik zum effizienten Einsatz von Sprachsteuerung M. Norda, J.-E. Appell, S.C. Lange, A. Hahn Sinkende Losgrößen, kürzere Produktlebenszyklen und die Hyper-Individualisierung von Produkten erschweren die wirtschaftliche Automatisierung von Produktionsprozessen, wodurch nutzerzentrierte und damit effiziente Human-MachineInterfaces wie Sprachsteuerung an Bedeutung gewinnen. Die Einflussfaktoren auf deren Effizienz in der Produktion sind vielfältig und komplex, weshalb in diesem Beitrag eine Methodik zur systematischen Identifikation von Anwendungsszenarien für den effizienten Einsatz von Sprachsteuerung vorgestellt wird. Methodology for the efficient use of voice control Decreasing batch sizes, shorter product-life-cycles and hyperindividualization of products make it difficult to automate production processes economically, which is why user-centric and therefore efficient human-machine interfaces such as voice control are becoming increasingly important. The factors influencing their efficiency in production are diverse and complex, thus this article presents a methodology for the systematic identification of application scenarios for the efficient use of voice control. STICHWÖRTER Industrie 5.0, Mensch und Technik, Arbeitsorganisation 1 Einleitung Die Anforderungen des globalen Marktes verändern sich hin zu immer individuelleren Produkten mit immer kürzerer Lebensdauer. Gleichzeitig beschleunigt die Entwicklungsgeschwindigkeit von neuen Technologien diesen Trend. Sinkende Losgrößen, kürzere Produktlebenszyklen und eine Hyper-Individualisierung von Produkten erschweren die wirtschaftliche Automatisierung von Produktionsprozessen. Die Flexibilität, mit der sich Produktionssysteme an die Anforderungen globaler Kunden anpassen können, wird in Zukunft ein entscheidender Wettbewerbsfaktor sein. [1–3] Industrie 5.0 greift die ursprünglichen Industrie 4.0-Ansätze zu Digitalisierung und KI-Technologien zur Steigerung der Effizienz und Flexibilität der Produktion auf und schafft einen ganzheitlichen Ansatz, der neben der Technologie im Rahmen eines nutzerzentrierten Ansatzes (engl.: human-centric approach) den Menschen mit einschließt. [1] Dabei werden die Intelligenz und Auffassungsgabe des Menschen mit all‘ seinen Sinnen in seiner neuen Funktion als Supervisor mit der Effizienz, künstlichen Intelligenz und Präzision von Maschinen kombiniert. Ziel ist es, Produktionsabläufen mit hohen Anforderungen an die Flexibilität, für die eine (vollständige) Automatisierung unwirtschaftlich ist, mit reduzierten Maschinenbedienzeiten und dadurch höherer Produktivität durch nutzerzentrierte Human-Machine-Interfaces (HMI) zu begegnen. [4, 5] Sprache bietet als natürlichste Form der Kommunikation das Potenzial, komplexe Menüstrukturen mit direkten Befehlen zu durchbrechen, Laufwege zu reduzieren und eine intuitive Mehr- 66 maschinenbedienung zu ermöglichen. [6, 7] Technisch sind Sprachsteuerungen, nicht zuletzt aufgrund der jüngsten Entwicklungen im Bereich künstliche Intelligenz (KI), hinsichtlich ihrer Robustheit auf einem für die Produktion nutzbaren Niveau. 2 Stand der Forschung und Motivation Sprachbefehle sind im Auto, in Mobiltelefonen sowie im Smart-Home-Bereich bereits als Standardschnittstelle etabliert. [6, 8, 9] Obwohl alle diese Sprachsteuerungen auf dem Prinzip der automatischen Spracherkennung (engl.: automatic speech recognition) (ASR) basieren, unterscheiden sich die Beweggründe zum Einsatz dieser. [10] Die Vorteile, wie freie Hände während der Interaktion, der Fokus auf eine Haupttätigkeit oder das Durchbrechen komplexer Menüstrukturen auf kleinen Bildschirmen helfen auch in der Produktion, jedoch ist der treibende Faktor für die Implementierung einer Sprachsteuerung in der Produktion eine potenzielle Effizienzsteigerung des Produktionsablaufs. [11, 12] Gayathri et al. verknüpften 2022 ein Sprachsteuerungsmodul (Elechouse V3) über einen Arduino-Microcontroller mit einer dreiachsigen Miniatur-CNC-Fräsmaschine, um zum einen die technische Funktion der Verknüpfung von Sprachsteuerung und CNC-Steuerung nachzuweisen und zum anderen die Vorteile einer Maschinenbedienung mit freien Händen zu untersuchen. [13] Schulte et al. implementierten 2021 eine SpracherkennungsAPI (Vosk/Alphacepei) in einen manuellen Montagearbeitsplatz, um die Betriebsdatenerfassung als nicht wertschöpfende Tätigkeit https://doi.org/10.37544/1436-4980-2024-03-38, am 14.07.2024, 02:22:22 Open Access – - https://elibrary.vdi-verlag.de/agb WT WERKSTATTSTECHNIK BD. 114 (2024) NR. 3 T I T E LT H E M A – FACHAUF SA T Z zu parallelisieren und damit die Effizienz von Montageprozessen zu steigern. [11] Gross et al. wiesen bereits 2020 mihilfe einer ConsumerSprachsteuerung (Amazon Echo Dot) nach, dass die grundsätzliche Steuerung eines Arduino-Roboters per Sprache einfach möglich ist. Gleichzeitig postulierten sie, dass weniger das konkrete Spracherkennungssystem, sondern mehr klare Gestaltungsregeln von Sprachbefehlen für die Interaktion zwischen Mensch und Maschine einen Einfluss auf die Gebrauchstauglichkeit (engl. Usability) des HMI und damit die Effizienz der Interaktion haben. [12] Trotz des technischen Reifegrads von Sprachsteuerung bleibt die flächendeckende Integration von Sprachsteuerung in der Produktion bisher aus. Neben der robusten Spracherkennungstechnologie sowie Adaptionsmöglichkeit an moderne industrielle speicherprogrammierbare Steuerungen (SPS) bedarf es Methoden zur gebrauchstauglichen und damit effizienten Implementierung von Sprachsteuerung in der Produktion, um das Potenzial sichtbar zu machen und einen Market-Pull zu generieren. 3 Einflussfaktoren auf die Effizienz von Sprachsteuerung als industrielles HMI Die Effizienz wird nach DIN EN ISO 9241–11 neben der Effektivität und Zufriedenstellung des Bedieners als Bestandteil der Gebrauchstauglichkeit von Mensch-Maschine-Systemen über das Verhältnis zwischen eingesetzten Ressourcen (Zeit, menschlicher Aufwand, Geld und Materialien) und den erreichten Ergebnissen definiert. Wesentliche Ressource für die Messung der Effizienz von HMIs ist die Zeit für die Ausführung einer Aufgabe, inklusive Korrektur etwaiger Eingabefehler. [14] Dabei hängt die Effizienz von HMIs zum einen von der Leistungsfähigkeit der Maschinenbediener und zum anderen von der Leistungsfähigkeit der jeweiligen HMI-Modalität ab. Die Leistungsfähigkeit der Maschinenbediener setzt sich aus vielen individuellen und zum Teil tagesformabhängigen Faktoren zusammen. Neben eher konstanten Faktoren, wie dem Ausbildungsgrad, speziellem Prozess- und Maschinenwissen sowie motorischen und visuellen Fähigkeiten, haben tagesindividuelle Faktoren, wie die Motivation und physische sowie kognitive Verfassung eines Maschinenbedieners, einen wesentlichen Einfluss auf die Interaktionsgeschwindigkeit und damit auf die Effizienz eines HMI. Da diese Faktoren, vor allem in Zeiten des Fachkräftemangels, immer schwerer beeinflussbar werden und gleichzeitig zu großen Teilen HMI-unabhängig sind, liegt der Fokus in diesem Beitrag auf den technischen Einflussfaktoren unter der Voraussetzung gleichbleibender menschlicher Einflussfaktoren im Betrachtungszeitraum. Die technischen Einflussfaktoren auf die Effizienz von Sprachsteuerung in der Produktion im Verhältnis zu konventionellen HMI, wie Knöpfen, Hebeln, Tastatur, Maus und Touchscreen sind vielfältig und komplex. Sie reichen von der Komplexität der Bedienungsaufgabe (Einschalten einer Prozesskammerbeleuchtung vs. Setzen eines neuen Werkstücknullpunkts) über das Befehlsdesign (Auswahl und Anordnung von Hotwords, Parametern und Attributen im Sprachbefehlssatz) und die Befehlskomplexität (Menüebenen und Anzahl an Interaktionen im konventionellen visuellen HMI) bis hin zur Bedienungsart (Automatikbetrieb, Teilautomatikbetrieb, Handbetrieb) der Maschine. Gleichzeitig spielen der Produktionsablauf mit seinen potenziellen Laufwegen, die Anzahl der zu bedienenden Maschinen sowie die Auswahl der WT WERKSTATTSTECHNIK BD. 114 (2024) NR. 3 Bild. Methodik zur Abschätzung des Effizienzpotenzials von Sprachsteuerung in der Produktion. Grafik: Fraunhofer IDMT https://doi.org/10.37544/1436-4980-2024-03-38, am 14.07.2024, 02:22:22 Open Access – - https://elibrary.vdi-verlag.de/agb 67 T I T E LT H E M A – FACHAUFSATZ Tabelle 1. Zeitäquivalente für Menüebenen [10] Menüebenen Zeitäquivalent 0 1,38 1 0,82 3 0,40 5 0,35 Tabelle 2. Zeitäquivalente für die Anzahl an Interaktionen [10] Interaktionen Zeitäquivalent 1 1,38 3 0,79 5 0,48 6 0,39 7 0,33 10 0,34 Audioschnittstelle (Headset, Mikrofon oder Mikrofon-Array) und deren Positionierung in der Produktionsumgebung eine wichtige Rolle. Nicht alle Einflussfaktoren haben in jedem Produktionsablauf den gleichen Einfluss auf die Effizienz der Kollaboration zwischen Mensch und Maschine, weshalb eine Quantifizierung von Einflussfaktoren sinnvoll ist. 4 Methodik zur Identifikation von Anwendungsszenarien für effiziente Sprachsteuerung Im Bild wird eine Methodik zur systematischen Abschätzung des Effizienzpotenzials von Sprachsteuerung in der Produktion auf Basis der drei übergeordneten Einflussfaktoren „Anzahl der zu bedienenden Maschinen“, „Befehlskomplexität in einem konventionellen HMI auf Basis von Menüebenen und Interaktionen“ sowie „Laufwegen im Produktionsprozess“ dargestellt. Die Beschränkung auf die drei Einflussfaktoren basiert auf der Abwägung zwischen der Komplexität der Methodik und der Präzision der Ergebnisse. Die detaillierteste Methodik, die alle Einflussfaktoren für die Abschätzung des Effizienzpotenzials umfasst, wird in der Praxis nicht angewendet. Mit dieser allgemeingültigen produktionsprozessunabhängigen Methodik kann bereits vor der Implementierung einer Sprachsteuerung die Effizienzsteigerung im Vergleich zu einem konventionellen HMI abgeschätzt werden. Im Schritt #1 wird die Anzahl an Maschinen identifiziert, die ein Bediener im Rahmen eines Arbeitstages bedient, und anschließend die genutzten Befehle pro Maschine entlang des Produktionsablaufs annotiert. Dies kann händisch durch ein Begleiten des Maschinenbedieners durch eine weitere Person (engl. Schadowing) oder aber automatisiert durch eine maschinenseitige Protokollierung geschehen. [15] Die Anzahl der Maschinen skaliert potenziell reduzierte Eingabezeiten in komplexen Menüstrukturen als Folge direkter Sprachbefehle oder die Vermeidung von Laufwegen durch Sprachbefehle. Im Folgenden wird im Schritt #2 die Komplexität jedes annotierten Befehls auf Menüebenen und die Anzahl der Interaktionen 68 im Menü des konventionellen grafischen HMI heruntergebrochen. Die Detaillierung über die Anzahl der Interaktionen ist notwendig, da auch auf einer Menüebene mehrere Interaktionen stattfinden können, wenn zum Beispiel gescrollt oder ein Eingabefeld ausgefüllt werden muss. Die Anzahl der Interaktionen ist also nur eine feinere Messgröße als die Anzahl der Menüebenen für die Komplexität des Graphical User Interface (GUI). [10] Nun werden im Schritt #3 die Eingabezeiten für jeden annotierten Befehl mit der konventionellen HMI-Modalität gemessen. Dieser Schritt ist schon schwieriger, da der Beginn der Interaktion mit der Maschine in einem konventionellen Maschinenprotokoll nicht automatisch erfasst wird, sondern lediglich der Zeitpunkt der letztendlichen Ausführung des Maschinenbefehls. Hier ist es spätestens notwendig, dass eine weitere Person mit einer Stoppuhr die Interaktionszeiten pro Befehl, im Besonderen aber den Start der Interaktion, erfasst. Jetzt werden im Schritt #4 die zugehörigen Laufwege (wenn vorhanden) pro Maschinenbefehl in Metern gemessen. Bewegt sich der Maschinenbediener während des Arbeitstages im Rahmen des Produktionsablaufs nicht zwischen mehreren Maschinen, so entfällt dieser Schritt. Läuft der Maschinenbediener zur Maschine und gibt mehrere Befehle nacheinander ein, so wird nur der erste Befehl mit einem Laufweg versehen. Nachdem die Länge der Laufwege ermittelt wurde, muss diese in die Laufzeit umgerechnet werden. Hierfür werden die tatsächlichen Laufzeiten der Maschinenbediener pro Laufweg per Stoppuhr erfasst. Die zweite Möglichkeit ist die Messung der durchschnittlichen Laufgeschwindigkeit von Bedienern im eigenen Unternehmen und das anschließende Dividieren der einzelnen Laufwege durch die durchschnittliche Laufgeschwindigkeit. Die dritte Möglichkeit ist die Nutzung des Methods-Time Measurement Universal Analysis System (MTM-UAS) der MTM Association e.V., laut der Mitarbeitende im Durchschnitt 25 Time-Measurement-Units (TMU), also 0,9 s benötigen, um einen Meter Laufweg zurückzulegen. Dies entspricht einer Laufgeschwindigkeit von 1,11 m/s. [16–18] Die dritte Methode, also die Verwendung eines empirisch ermittelten Durchschnitts, ist für die Abschätzung des Effizienzpotenzials von Sprachsteuerung in dieser Methode für die Genauigkeit ausreichend. Im folgenden Schritt #5 werden die Interaktionszeiten des konventionellen HMI für jeden Befehl, entsprechend seiner Menüebene, beziehungsweise der Anzahl an Interaktionen mit den Zeit-Äquivalenten für einen entsprechenden Sprachbefehl multipliziert, um die Interaktionszeit für eine Sprachsteuerung zu erhalten. Sprachbefehle erlauben die direkte Adressierung von Maschinenbefehlen, die konventionell nur zeitintensiv über mehrere Menüebenen und Interaktionen erreichbar sind. In Tabelle 1 und Tabelle 2 werden exemplarisch Zeitäquivalente für die Einsparung von Menüebenen und Interaktionen durch Sprachsteuerung im Vergleich zu einer Touchscreensteuerung gezeigt. Diese wurden in einer Probandenstudie, bei der ein stateof-the-art (SOTA) GUI mit einer Sprachsteuerung verglichen wurde, empirisch ermittelt. Zeitäquivalente größer eins zeigen längere Eingabezeiten und Zeitäquivalente unter eins kürzere Eingabezeiten durch Sprachsteuerung an. [10] Nachfolgend werden im Schritt #6 die durch eine Sprachsteuerung vermeidbaren Laufwege identifiziert. Im Kontext der Effizienz von HMIs lassen sich die Laufwege vier Kategorien zuordnen. Zu den „unvermeidbaren“ Laufwegen zählen Wartungs-, Bedienungs- und Handhabungstätigkeiten, die sich nicht über die https://doi.org/10.37544/1436-4980-2024-03-38, am 14.07.2024, 02:22:22 Open Access – - https://elibrary.vdi-verlag.de/agb WT WERKSTATTSTECHNIK BD. 114 (2024) NR. 3 T I T E LT H E M A Arbeitsorganisation optimieren lassen und/oder unabhängig von der HMI-Modalität sind. Laufwege, die sich durch eine „optimierte Arbeitsorganisation“ vermeiden lassen, sind beispielsweise Laufwege in der Produktionshalle, um benötigte Werkzeuge und Materialien zur Störungsbeseitigung zu beschaffen, die auch bereits vorkonfektioniert am Arbeitsplatz zur Verfügung stehen könnten. Durch ein „optimiertes HMI-Konzept (ohne Sprachsteuerung)“, wie einen zentralen Leitstand, zentralen Bedienpunkte oder mobile Endgeräte können ebenfalls Laufwege in der Produktion eingespart werden. Für die Methodik sind nur Laufwege der letzten Kategorie, die „durch Sprachsteuerung vermeidbar“ sind, relevant. Mithilfe einer Sprachsteuerung können Bediener beispielsweise über ein Mikrofon-Array mit freien Händen einen Roboter einrichten, ohne Laufwege zwischen dem Endeffektor und dem HMI zurücklegen zu müssen. Gleichzeitig können sie Laufwege vermeiden, indem sie über ein Headset ortsunabhängig den Status mehrerer Maschinen abfragen, neue Halbzeuge per Automated Guide Vehicle (AGV) ordern oder nächste Prozessschritte in eigensicheren Maschinen starten. Die Kategorisierung der verschiedenen Arten von Laufwegen in der Produktion wurde in einer Industrieuntersuchung mit mehreren deutschen Unternehmen entwickelt. [16] Im letzten Schritt #7 werden über alle Maschinen des Bedieners die Eingabezeiten sowie die zugehörigen Laufwegzeiten des konventionellen HMIs aufsummiert und durch die Eingabezeiten und zugehörigen Laufwegzeiten einer potenziellen Sprachsteuerung dividiert. Zuletzt wird der Quotient in Prozent umgerechnet. Negative Prozentwerte zeigen eine verschlechterte Effizienz und positive Prozentwerte eine gesteigerte Effizienz der Interaktion durch den Einsatz einer Sprachsteuerung an. 5 Fazit und Ausblick FACHAUF SA T Z Produktionsabläufe und zugehöriger konventioneller HMIs. Dies ist besonders im Bereich der GUIs von Produktionsmaschinen und Laufwegen, die durch eine optimierte Arbeitsorganisation oder ein optimiertes HMI-Konzept vermieden werden können, der Fall. FÖRDERHINWEIS Diese Arbeit wurde im Rahmen des Projekts „Voice-Controlled Production“ (VCP) am Fraunhofer-Institut für Digitale Medientechnologie IDMT, Institutsteil Oldenburg für Hör-, Sprach- und Audiotechnologie HSA, in Kooperation mit der Hochschule Emden/Leer durchgeführt. Das Fraunhofer IDMT in Oldenburg wird im Rahmen des Programms „Vorab“ durch das Niedersächsische Ministerium für Wissenschaft und Kultur und durch die Volkswagen Stiftung für seine Weiterentwicklung gefördert (Aktenzeichen 11 – 76251–2005/2021 (ZN3812)). DANKSAGUNG Vielen Dank an unsere Kollegen vom Fraunhofer IDMT in Oldenburg, Jens Adrian und Matthias Stennes, für das stets kritische Feedback und die Unterstützung bei der Aufbereitung der Forschungsergebnisse sowie Entwicklung der vorgestellten Methodik. Ebenfalls einen herzlichen Dank an Lorenz Arnold, Geschäftsführer der Firma MGA-Ingenieurdienstleistungen GmbH aus Würzburg, der das Projekt „Voice-Controlled Pro- Die entwickelte Methodik senkt für produzierende Unternehmen die Hürde für die Implementierung neuartiger, nutzerzentrierter HMIs, wie zum Beispiel einer Sprachsteuerung, da bereits im Vorhinein eine potenzielle Effizienzsteigerung abgeschätzt wird. Die systematische Analyse des Produktionsablaufs entlang der Methodik wird ohne den Eingriff in bestehende Hard- oder Software, wie es bei der Alternative, der tatsächlichen Integration einer Sprachsteuerung als weitere HMI-Modalität an der Maschine und dem anschließenden Vergleich von Eingabezeiten und Laufwegen der Fall wäre, durchgeführt. Mit überschaubarem Aufwand wird so zum Teil aus bestehenden Daten das produktionsablaufspezifische Potenzial zur Effizienzsteigerung durch eine Sprachsteuerung abgeschätzt. Der tatsächliche Vergleich der realen Bedienzeiten einer Sprachsteuerung mit einem konventionellen HMI desselben Produktionsablaufs, Bedieners, Maschinen, Befehlen und Laufwegen wird immer die präziseren Messwerte hervorbringen als die Abschätzung des Effizienzpotenzials mit Hilfe empirisch ermittelter Zeitäquivalente und berechneten Laufzeiten über TMUs. Der Aufwand zur Integration einer Sprachsteuerung ist aber gleichzeitig auch immer höher als die Identifikation der effizientesten HMI-Modalität mit der Methodik. Neben der Abschätzung des Effizienzpotenzials einer Sprachsteuerung führt die systematische Auseinandersetzung mit HMIs von Produktionsmaschinen auch zu Synergieeffekten bei der Identifizierung verborgener Optimierungspotenziale vorhandener WT WERKSTATTSTECHNIK BD. 114 (2024) NR. 3 – duction“ von Anfang an als Industrie-Gutachter begleitet und uns nie die Nähe zur industriellen Praxis verlieren lassen hat. L i tera tu r [1] Breque, M.; Nul, L. de; Petridis, A.: Industry 5.0. Towards a sustainable, human-centric and resilient European industry. Luxembourg: Publications Office of the European Union 2021 [2] Khan, M.; Haleem, A.; Javaid, M.: Changes and improvements in Industry 5.0: A strategic approach to overcome the challenges of Industry 4.0. 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A p pe l l Fraunhofer-Institut für Digitale Medientechnologie IDMT Institutsteil Hör-, Sprach- und Audiotechnologie HSA Marie-Curie-Str. 2, 26129 Oldenburg P ro f. D r. - I n g . L a nge Sve n C a r s ten Hochschule Emden/Leer Fachbereicht Technik, Abteilung Maschinenbau Constantiaplatz 4, 26723 Emden P rof. D r. - I n g . A xe l H a h n Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR) Institut Systems Engineering für zukünftige Mobilität Carl-von-Ossietzky-Universität Oldenburg Escherweg 2, 26121 Oldenburg LIZENZ Dieser Fachaufsatz steht unter der Lizenz Creative Commons Namensnennung 4.0 International (CC BY 4.0) 70 https://doi.org/10.37544/1436-4980-2024-03-38, am 14.07.2024, 02:22:22 Open Access – - https://elibrary.vdi-verlag.de/agb WT WERKSTATTSTECHNIK BD. 114 (2024) NR. 3
https://openalex.org/W2416876620	https://europepmc.org/articles/pmc4896969?pdf=render	English	null	Design of a high-mobility multi-terrain robot based on eccentric paddle mechanism	Robotics and biomimetics	2,016	cc-by	8,405	© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Gaining high mobility on versatile terrains is a crucial target for designing a mobile robot toward tasks such as search and rescue, scientific exploration, and environment monitoring. Inspired by dextrous limb motion of animals, a novel form of locomotion has been established in our previous study, by proposing an eccentric paddle mechanism (ePad- dle) for integrating paddling motion into a traditional wheeled mechanism. In this paper, prototypes of an ePaddle mechanism and an ePaddle-based quadruped robot are presented. Several locomotion modes, including wheeled rolling, legged crawling, legged race-walking, rotational paddling, oscillating paddling, and paddle-aided rolling, are experimentally verified on testbeds with fabricated prototypes. Experimental results confirm that paddle’s motion is useful in all the locomotion modes. Keywords: Eccentric paddle mechanism, ePaddle, Multi-terrain locomotion Design of a high‑mobility multi‑terrain robot based on eccentric paddle mechanism Yi Sun1, Yang Yang1,2, Shugen Ma1* and Huayan Pu3 Sun et al. Robot. Biomim. (2016) 3:8 DOI 10.1186/s40638-016-0041-3 Open Access Background separated legs and wheels that can behave as a legged robot and a wheeled robot cooperatively and simulta- neously. For instance, Chariot-II constructed at Tohoku University (Japan) has two big active wheels at the both sides of the body and four legs with three joints at the both ends of the body [9]. ALDURO proposed by Hiller et al. [10] has wheels on the back legs but no wheels on the front legs. Wheeleg [11] has two front legs, each with three-DOF prismatic joints, and two independently actu- ated back wheels. As learned from realistic search and rescue activities at disaster sites, the mobility of the mobile robot is of the greatest importance [1]. To guarantee its mobility, the locomotion mechanism of the robot should be able to adapt to diverse terrestrial, aquatic, and amphibious ter- rains. Robots based on single locomotive forms lack such crucial multi-terrain adaptivity which prevents them from being practically used in realistic search and rescue activities. Robots in the second group have kinematically syn- thesized locomotion mechanisms which are multi-func- tional. For example, Hong et al. proposed a mechanism consisting of two rimless wheels with individually actu- ated spokes in Intelligent Mobility Platform with Active Spoke System (IMPASS) to travel on uneven surfaces like tracks and step over obstacles like legged vehicles while retaining the simplicity of wheels [12]. Perpendic- ularly Oriented Planetary LEgged Robot (PEOPLER-II) proposed by Okada et al. [13] uses pairs of legs jointed at a wheel rim so that the leg can fold in walking and unfold in rolling. Steffan et al. presented the idea of cel- lular locomotion mechanism in [14], where the proposed mechanism generates rectilinear locomotion exclusively through linear actuation of the legs. In [15], the authors discussed a rimless wheel with radially expanding legs, Recently, hybrid locomotion mechanisms have been proposed toward multi-terrain mobility for mobile robot. Most of them are designated for terrestrial-task-oriented robots, which consist of the following two major groups. The first group includes those robots that are composites by basic motion units such as wheels and legs or those units that act together to provide locomotion. Most of them are articulated-wheeled robots which have wheels placed at the end of the legs, including Shrimp [2], Octo- pus [3], ATHLETE [4], Hylos [5], Roller-Walker [6], AZIMUT [7] and Robot IMR-Type I [8]. Correspondence: shugen.ma@ieee.org 1 Department of Robotics, Ristumeikan University, 1‑1‑1 Noji Higashi, Kusatsu, Shiga 525‑8577, Japan Full list of author information is available at the end of the article Concept of the eccentric paddle mechanism b Schematic drawing of the ePaddle mechanism b Sun Gear Paddle-shaft L L2 Rs RH 1 R Wheel-rim-hinge Paddle Planetary Gear Carrier Paddle Wheel-rim-hing Wheel Paddle-shaft Sun Gear Planetary Gear Carrier a Concept of the eccentric paddle mechanism Legged locomotion modes, such as running, climbing, and paddling, bring remarkable multi-terrain adaptivity to animals. However, wheeled motion is superior to leg- ged one in speed and energetic efficiency. The best way to acquire multi-terrain adaptivity, speed, and energetic efficiency for an mobile robot is finding a method to inte- grate legged motion into wheeled locomotion. However, limited by their locomotion ability, above amphibious robots still cannot be applied for search and rescue tasks after tsunami, where multiple gaits on ter- restrial, aquatic, and amphibious terrains are required.f Based on this idea, concept of the eccentric paddle mechanism (ePaddle), as shown in Fig. 1, has been pro- posed firstly in [30]. The ePaddle mechanism consists of a wheeled shell, a set of rigid paddles, a paddle-shaft, pad- dle-hinges, and wheel-rim-hinges. We start our effort to develop an amphibious robot that possesses high mobility and high energetic efficiency in complex amphibious environment by proposing a novel locomotion mechanism: eccentric paddle mechanism (ePaddle) [29]. The concept of the ePaddle is shown in Fig. 1. By actively locating the paddle-shaft inside the wheeled shell via independent actuators, motion pat- terns of the ePaddle can be alternated. Five locomotion gaits (as shown in Fig. 2) have been proposed in our pre- vious work, three of which are terrestrial gaits, namely the wheeled rolling gait [30], the legged walking gait [31, 32], and the legged-wheeled hybrid gait [33, 34]; and two of which are aquatic gaits, named the rotational paddling A wheeled shell of the ePaddle module is actuated by a motor to rotate around the wheel axis, and a set of pad- dles are mounted on and can passively rotate around a paddle-shaft inside the wheel. Wheel-rim-hinges placed on the wheel’s rim hold the paddles and allow the paddles to passively slide through them. Location of the paddle- shaft with respect to the wheel axis is actively positioned by two motors via a planetary gear train. By tuning paddles’ motion, versatility in locomotion modes is brought to the ePaddle mechanism. Figure 2 demonstrates several examples of paddle-aided motions that are feasible for an ePaddle-based robot. Paddle Wheel-rim-hing Wheel Paddle-shaft Sun Gear Planetary Gear Carrier b a Sun Gear Paddle-shaft L L2 Rs RH 1 R Wheel-rim-hinge Paddle Planetary Gear Carrier Fig. 1 Concept of the eccentric paddle mechanism. a Major com- ponents in an ePaddle module. Background Others have Correspondence: shugen.ma@ieee.org 1 Department of Robotics, Ristumeikan University, 1‑1‑1 Noji Higashi, Kusatsu, Shiga 525‑8577, Japan Full list of author information is available at the end of the article Sun et al. Robot. Biomim. (2016) 3:8 Page 2 of 11 gait [35] and the oscillating paddling gait [36, 37]. The ability to generate thrusts for underwater propulsion has been observed as well [36, 37]. intending to travel over obstacles by expanding the wheel instead of avoiding obstacles. A bipedal robot that com- bines the rolling, walking, and climbing locomotion was proposed by Shores et al. [16]. Lin et al. proposed a wheel-leg hybrid robot that can change the morphology of full-circle wheels into half-circle legs using a trans- formation mechanism [17, 18]. To change locomotion mode, robots in this category usually need performing shape alternating procedures. Based on our preliminary analysis or experiments on locomotion modes of an ePaddle mechanism, interesting findings relative to how paddle’s motion could improve environmental adaptability have been noticed. In this paper, the effects of paddle’s motion on multi-terrain locomotion are focused on. Prototypes of the ePaddle module and an ePaddle-based quadruped robot are fabri- cated for experimental verification. Experimental results show that paddle-aided motion is useful for generating vectored thrusts in aquatic environment, for improving soil reaction forces on soft sandy terrain, and for enhanc- ing obstacle-negotiating capability on rough terrains. Several amphibious robots are based on wheels [19] and tracks [20]. Some robots are inspired from natural creatures, such as robot turtle by Low et al. [21], crab-like robot by Wang et al. [22], lobster by Ayers et al. [23], and snake-like robot AmphiBot I and II by Crespi et al. [24, 25]. We also have developed an amphibious snake-like robot [26]. Other robots with novel hybrid mechanisms have been reported as well. For instance, an autonomous amphibious vehicle which can travel on aquatic and ter- restrial terrains by using four driven paddle wheels was presented by Frejek et al. [27]. Dubbed Whegs™ IV [28], which is based on Whegs™, has six wheel legs and is capable of walking in a tripod legged gait and swimming underwater. Kinematics of the ePaddle mechanism The wheeled shell, as shown in Fig. 3c, is constructed from two annulus frames, eight shell pieces, and eight wheel-rim-hinges. The shells are fixed between the Kinematics of the ePaddle mechanism describes kin- ematic relationship between the lug’s motion and joint’s motion. As shown in Fig. 4a, a coordinate system named wheel coordinates is defined in the way that the origins of the coordinates locate at the wheel center, and the x-axis is in the horizontal direction. Posture of the ePaddle, or in other word, positions and orientations of ePaddle’s components with respect to the wheel coordinates, is a Paddle-shaft Lug Wheel b c Wheel Rim Wheel-rim-hing Paddle Paddle-shaft Bearing Gear Copouling Motor Carrier Sun Gear Planetary Gear Motors Absolute Encoders Fig. 3 Prototype design of the ePaddle module. a Front view and b rear view of an assembled prototype module. c Section view of the CAD drawing of the module a P ft b Motors Absolute Encoders Table 1 Specifications of the ePaddle prototype module Item Description Paddle Dimensions Length, L: 96.5 mm; width: 63 mm; thickness: 2 mm Moving range RS = LA + LB = 40 mm, where LA = LB Material Aluminum alloy (A5052) Weight 36 g (eight lugs with eight paddle-hinges) Wheel shell Dimensions Outer radius, RA: 56.5 mm; width: 113 mm; Material Aluminum alloy (A5052) Wheel-rim-hinges Dimensions Pitch circle radius, RH: 50 mm Materials Aluminum alloy (A5052) Assembled module Weight 3.7 kg (with three motors) Table 1 Specifications of the ePaddle prototype module Item Description Paddle Dimensions Length, L: 96.5 mm; width: 63 mm; thickness: 2 mm Moving range RS = LA + LB = 40 mm, where LA = LB Material Aluminum alloy (A5052) Weight 36 g (eight lugs with eight paddle-hinges) Wheel shell Dimensions Outer radius, RA: 56.5 mm; width: 113 mm; Material Aluminum alloy (A5052) Wheel-rim-hinges Dimensions Pitch circle radius, RH: 50 mm Materials Aluminum alloy (A5052) Assembled module Weight 3.7 kg (with three motors) b a a b c Wheel Rim Wheel-rim-hing Paddle Paddle-shaft Bearing Gear Copouling Motor Carrier Sun Gear Planetary Gear c Bearin Fig. 3 Prototype design of the ePaddle module. a Front view and b rear view of an assembled prototype module. c Section view of the CAD drawing of the module Fig. 3 Prototype design of the ePaddle module. a Front view and b rear view of an assembled prototype module. Design of the ePaddle‑based prototype robot Prototype ePaddle moduleh Specifications of the ePaddle module are listed in Table 1. Design of the ePaddle‑based prototype robot Prototype ePaddle moduleh The fabricated ePaddle prototype module is shown in Fig. 3a. Key components, such as the wheel-rim-hinge, the paddle-hinge, and the transmission chains of the pro- totype module, are shown in Fig. 3b, c. Planetary Gear As shown in Fig. 3c, the wheel rim, carrier, and sun gear are separately driven by three DC brushed motors [Maxon RE25, integrated with a GP gearbox (72:1)] via gears. The angular positions of the three joints are b a a Fig. 1 Concept of the eccentric paddle mechanism. a Major com- ponents in an ePaddle module. b Schematic drawing of the ePaddle mechanism Fig. 1 Concept of the eccentric paddle mechanism. a Major com- ponents in an ePaddle module. b Schematic drawing of the ePaddle mechanism Sun et al. Robot. Biomim. (2016) 3:8 Page 3 of 11 d n a L er o h S cita u q A er o h S d n a L Man-Constructed Terrains Natural Terrains Wheeled Gait Wheel-Leg-Integrated Gait Wheel-Leg-Integrated Gait Paddling Gait Legged Gait Legged Gait Wheeled Gait Fig. 2 Multi-terrain locomotion modes. An ePaddle-based robot is capable of performing versatile gaits in complex environments [30] cita u q A d n a L Wheel-Leg-Integrated Gait Wheel-Leg-Integrated Gait collected by three absolute encoders (RE22, RLS; Slove- nia). The wheel rim is rotated by the first motor through a pair of flat gears. The position of the paddle-shaft is determined by the angular position of the carrier and sun gear of planetary gear mechanism.hi frames by stainless steel pins and bolts to form the wheel surface. The wheel-rim-hinges, which freely rotate around their central axis, are supported between the frames by the bearings. The paddle slides through a hole inserted in the wheel-rim-hinge.i For dust-proofing, the clearances between the paddle and the wheel-rim-hinge and between the wheel-rim- hinge and the shell are filled with woolen felt sheets. The outer surface of the wheel is polished for future adhesion of rubber tire.i The stainless steel paddle-shaft is fixed to the disk integrated with the planetary gear by pins and bolts. As shown in Fig. 3c, the paddles are made from rectangular rigid aluminum plates, and each paddle is hung on the paddle-shaft by a paddle-hinge. Ball bearings are assem- bled in the paddle-hinge, ensuring smooth passive rota- tion of the paddle around the paddle-shaft.h Specifications of the ePaddle module are listed in Table 1. Wheeled rolling mode A wheeled rolling motion can be performed by making the outer surfaces of the shells in contact with ground and roll- ing as wheels. The paddle-shaft is located at a proper posi- tion to pretend the paddles punching with the ground. Kinematics of the ePaddle mechanism c Section view of the CAD drawing of the module Sun et al. Robot. Biomim. (2016) 3:8 Page 4 of 11 On-board PC Controllers Ground Front-module Rear-module Fig. 5 Prototype of an ePaddle-based quadruped robot. Four ePad- dle modules are mounted on the robot body where a network of custom-designed real-time controllers and an onboard computer are installed. The robot can be powered by either a power tether or a onboard battery Paddle 5 Paddle 6 Paddle 7 b a Fig. 4 Kinematics of the ePaddle module. a Coordinates definitions. b Definitions of the joint angles (only one of the paddle is shown for better clarity) b a Fig. 4 Kinematics of the ePaddle module. a Coordinates definitions. b Definitions of the joint angles (only one of the paddle is shown for better clarity) Fig. 5 Prototype of an ePaddle-based quadruped robot. Four ePad- dle modules are mounted on the robot body where a network of custom-designed real-time controllers and an onboard computer are installed. The robot can be powered by either a power tether or a onboard battery determined by angular positions of the wheel joint, the base joint, and the arm joint, respectively, denoted by three joint parameters, θ0, θ1, and θ2. For example, as shown in Fig. 4b, inclination angle and tip position of paddle-1 can be, respectively, expressed by The real-time motion controller, as shown in Fig. 6a, based on a dsPIC33-FJ128MC804 microcontroller (Microchip, USA) is custom-designed in a highly recon- figurable way to enable easy reconfiguration of the con- troller network without major modifications in hardware. The schematic drawings of the motion controller and the controller network are shown in Fig. 6a, b, respectively. Figure 6c shows an assembled controller network for an ePaddle module. (1) θPi = atan 2(yHi −yS, xHi −xS) d (1) θPi = atan 2(yHi −yS, xHi −xS) (1) and and (2) xPi = xS + L cos θPi yPi = yS + L sin θPi (2) where S = [xS, yS]T and Hi = [xHi, yHi]T are the position of the paddle-shaft and the position of the wheel-rim- hinge-1, respectively, and L is the length of the paddle. They can be expressed by where S = [xS, yS]T and Hi = [xHi, yHi]T are the position of the paddle-shaft and the position of the wheel-rim- hinge-1, respectively, and L is the length of the paddle. They can be expressed by Multi‑terrain locomotion modesi (3) xS = L1 cos θ1 + L2 cos θ2 yS = L1 sin θ1 + L2 sin θ2 At current stage, five types of feasible locomotion modes have been found for an ePaddle-based robot, which are summarized in Fig. 2. In addition, a novel hybrid locomo- tion mode named Wheel-leg-integrated Mode (or Active Lugged Mode) is found to be helpful when the robot trav- els on soft terrains. (3) and and (4) xHi = RH cos θHi yHi = RH sin θHi (4) where θHi = θ0 + (i −1)π/2 is the posture angle of the wheel-rim-hinge-i; L1 and L2 are lengths of the base link and the arm link, respectively; RH is the radius of the cir- cular where the wheel-rim-hinges locate. ePaddle‑based quadruped robot To avoid paddle-ground-collision two critical require- ments should be fulfilled in designing the ePaddle mod- ule and planning its wheeled motion. The first one is that the shortest protruding length of the paddles should be zero, which means the following geometric constraint should be satisfied: An ePaddle-based quadruped robot equipped with four ePaddle modules has been assembled as shown in Fig. 5. A controller network consisting of multiple motor controllers and a commander computer is carried on body of the robot. A power tether is used for supplying power source to the robot for conducting indoor experi- ments, whereas an onboard battery is used for outdoor experiments. i (5) L ≤RS + RA L ≤RS + RA (5) Page 5 of 11 Sun et al. Robot. Biomim. (2016) 3:8 Fig. 6 Custom-designed reconfigurable motion controller. a Proto- type. b Schematic drawing of the controller. c Schematic drawing of the controller network a b c d Fig. 7 Locating paddle-shaft at a proper place for avoiding paddle- ground collision in wheeled mode [30]. a On even ground, b on a slope, c on the top of a slope, d on the bottom of a slope b c d a d b c a Fig. 7 Locating paddle-shaft at a proper place for avoiding paddle- ground collision in wheeled mode [30]. a On even ground, b on a slope, c on the top of a slope, d on the bottom of a slope oscillating paddling, can be performed by an ePaddle mechanism.hi The first type is the rotational paddling motion [35], as shown in Fig. 8a. During this gait, all the paddles are driven by the wheel to rotate in one direction around the paddle- shaft. Because the paddle-shaft is located eccentrically, the protruding areas of the paddles are different, which gener- ate different amount of thrusts. By alternating the location of the paddle-shaft and rotational speed of the wheel, we can change the magnitude and direction of the thrusts.h Fig. 6 Custom-designed reconfigurable motion controller. a Proto- type. b Schematic drawing of the controller. c Schematic drawing of the controller network The second aquatic motion, as shown in Fig. 8b, is the oscillating paddling motion [38]. For our ePaddle mechanism, we initially choose one of the four pad- dles in the ePaddle as the working paddle. The paddle- shaft is located at a position with an eccentric distance rS. ePaddle‑based quadruped robot The wheel-like shell is driven to rotate, but the rota- tional direction is periodically reversed. It will result in the oscillation of the working paddle at an average posi- tion Ai0 with an angular magnitude θOsc. The direction and magnitude of the generated thrusts are adjustable by changing Ai0 and θOsc, respectively. Fig. 6 Custom-designed reconfigurable motion controller. a Proto- type. b Schematic drawing of the controller. c Schematic drawing of the controller network Fig. 6 Custom-designed reconfigurable motion controller. a Proto- type. b Schematic drawing of the controller. c Schematic drawing of the controller network where RS = √(x2 S + y2 S) is the radius of the workspace of paddle-shaft and RA is the radius of the wheel. In our pro- totype, we let L = RS + RA for maximizing the workspace of the paddles.h To generate smooth oscillating motion, the angular position of the wheel is driven to follow sinusoidal-based trajectories defined as The second requirement is that the paddle-shaft should be located at a proper position for ensuring the land- ing paddle is totally retracted when the landing paddle touches the ground. As presented in [30], a practical choice of paddle-shaft’s position is the farthest posi- tion away from the contact point. Figure 7 shows several examples of selecting positions of paddle-shaft on variety of terrains.h (6) θW,sym = θOsc sin 2πt T + π 2 . (6) Legged walking modesh The paddles integrated in the ePaddle mechanism also have the ability to serve as legs on rough terrains in two different modes.hi The paddles integrated in the ePaddle mechanism also have the ability to serve as legs on rough terrains in two different modes.hi f The first type is the crawling motion mode that is simi- lar to the crawling gait of traditional open-link based leg- ged robots [39]. For a ePaddle-based quadruped robot working in this mode, three of the four legs are in contact with the ground to support the weight of the robot, and another one leg is swinging in the air. Detailed discussion on this mode has been presented in [30].h The advantage of the wheeled rolling mode is its sim- plicity in control. Once the paddle-shaft is properly located, only the wheel is actively actuated. Comparing with traditional wheeled rolling, the presence of the pad- dles improves obstacle-negotiating capability of the ePad- dle module by allowing the module to “hook” or “climb” on the obstacles. The second legged mode is the novel race-walking motion mode, which is a unique legged motion for the ePaddle mechanism. For an ePaddle-based quadruped robot working in this mode, all the four ePaddle modules Setups of locomotion experiments Aquatic paddling experiments are always in contact with the ground during the locomo- tion which results the robot is supported by at least four legs at any time. Planning method of this mode has been discussed in [31]. As shown in Fig. 10, a simplified ePaddle module equipped with four paddles was mounted on an alu- minum frame and submerged beneath the water surface of a water tank. A force/torque sensor holding the alu- minum frame was fixed on the supporting frame above the water surface for measuring the thrust forces gener- ated by the ePaddle module. Sampling rate of the meas- urement system was 1000 Hz. Aquatic paddling modes In an aquatic environment, two types of swimming motions, namely the rotational paddling and the Sun et al. Robot. Biomim. (2016) 3:8 Page 6 of 11 b a Fig. 8 Two feasible paddling modes of the ePaddle mechanism. a Rotational paddling motion. b Oscillating paddling motion Motor Controller F/T Sensor Motor Power Wheel Pos. Encoder (500Hz) RS232 Port DAQ-AD F/T Sensor Amplifier DAQ-DIO PC Force/Torque (500Hz) Motor Cmd. (Triggered) Motor Encoder (2000Hz) ePaddle Water Tank PC Motor Controller Wheel Paddle T-shaped Beam Motor Supporting Frame Supporting Frame a b x y z 120mm x y z Fig. 10 Testbed with a water tank for measuring aquatic thrust forces. a Assembled water tank. b Schematic drawing of the data acquisition system b a Fig. 8 Two feasible paddling modes of the ePaddle mechanism. a Rotational paddling motion. b Oscillating paddling motion Motor Controller F/T Sensor Motor Power Wheel Pos. Encoder (500Hz) RS232 Port DAQ-AD F/T Sensor Amplifier DAQ-DIO PC Force/Torque (500Hz) Motor Cmd. (Triggered) Motor Encoder (2000Hz) ePaddle Water Tank PC Motor Controller Wheel Paddle T-shaped Beam Motor Supporting Frame Supporting Frame a b x y z 120mm x y z Fig. 10 Testbed with a water tank for measuring aquatic thrust forces. a Assembled water tank. b Schematic drawing of the data acquisition system es of the ePaddle mechanism a b a Motor Controller F/T Sensor Motor Power Wheel Pos. Encoder (500Hz) RS232 Port DAQ-AD F/T Sensor Amplifier DAQ-DIO PC Force/Torque (500Hz) Motor Cmd. (Triggered) Motor Encoder (2000Hz) ePaddle Water Tank PC Motor Controller Wheel Paddle T-shaped Beam Motor Supporting Frame Supporting Frame a b x y z 120mm x y z Fig. 10 Testbed with a water tank for measuring aquatic thrust forces. a Assembled water tank. b Schematic drawing of the data acquisition system a b Fig. 10 Testbed with a water tank for measuring aquatic thrust forces. a Assembled water tank. b Schematic drawing of the data acquisition system Fig. 8 Two feasible paddling modes of the ePaddle mechanism. a Rotational paddling motion. b Oscillating paddling motion Active‑lugged mode (Wheel‑leg‑integraged mode) On sandy or muddy soft terrains, both legs and wheels may fail due to the limited contact areas and the slid- ing, respectively. An ePaddle-based robot can perform active-lugged locomotion mode by digging its paddles into the soil to generate additional supporting or track- ing forces. Sandy rolling experiments A testbed of length 1700 mm, width 500 mm, and height 800 mm was designed for conducting locomo- tion experiments on sandy terrain, as shown in Fig. 11. A linear conveyance unit was actuated at desired speeds by a DC brushed servo motor via a ball-bearing screw. A fully functional ePaddle module was mounted on the conveyance unit via a six-axis force/torque sensor (Delta SI-330-30, ATI; USA). Vertical position of the ePaddle module with respect to the ground surface was fixed during the experiments for eliminating influence of wheel’s sinkage on measured soil reaction forces. Standard sequence of the active-lugged locomotion, as shown in Fig. 9, consists of three phases: the soil-entering, bulldozing, and recovering phase, according to paddle’s motion. In all the phases, the wheel is driven to rotate at a constant speed, whereas the paddle-shaft is actively actu- ated to follow a predefined trajectory. Paddle-shaft’s tra- jectory is designed in a way that a desired planar motion of the paddle can be got in the bulldozing phase. Pure translational and pure rotational motions are two of the example planar motions of the paddle in the bulldozing phase. Fig. 9 Standard sequence of the active-lugged locomotion g. 9 Standard sequence of the active-lugged locomotion Page 7 of 11 Sun et al. Robot. Biomim. (2016) 3:8 Fig. 11 Sandy testbed for measuring soil-ePaddle reaction mechan- ics. a Assembled testbed. b Schematic drawing of the data acquisi- tion system an A/D board (AD12-16(PCI), Contec; Japan) at the same rate as the encoders. Soft dry sand filled in the sandbox of the testbed has been purified, sieved, ventilated, and dried. The physical and mechanical properties of the soil are listed in Table 2. Terrestrial walking experiments A single ePaddle prototype module was tested in several scenarios to verify the terrestrial locomotion. The tested scenarios include rolling on even ground, climbing up wood steps, ascending a wood slope of several inclination angles, and negotiating with stacks of wood poles. A quadruped prototype robot equipped with four ePaddle modules, as shown in Fig. 5, has been fabricated to verify the proposed controller network as well. Results and discussion Fig. 11 Sandy testbed for measuring soil-ePaddle reaction mechan- ics. a Assembled testbed. b Schematic drawing of the data acquisi- tion system ePaddle‑soil reaction mechanics in sandy terrain As shown in Fig. 14, the drawbar pull and vertical force of the ePaddle module were measured and compared with the results of a fixed lugged wheel that has the same radius as the ePaddle’s wheel. Generating vectored thrusts in aquatic environment 13 Thrusts generated in oscillating paddling mode. Measured thrusts in a x-direction and in b y-direction with different oscillation amplitudes. The oscillation period is 1.5 s and the paddle-shaft is placed at its outmost position along the x-axis the paddle-shaft at an eccentric position with respect to wheel center along the y-direction. It implies that vec- tored thrusts are achievable by tuning paddle-shaft’s position. 30 60 90 0 50 Angular position of the wheel [deg] Fig. 14 Soil-ePaddle interaction mechanics. Comparison of a draw- bar pulls and b vertical forces generated by a fixed lugged wheel (lug height = 13 mm) and by the ePaddle mechanism at various of sinkage lengths and 60° inclination angle Fig. 14 Soil-ePaddle interaction mechanics. Comparison of a draw- bar pulls and b vertical forces generated by a fixed lugged wheel (lug height = 13 mm) and by the ePaddle mechanism at various of sinkage lengths and 60° inclination angle Figure 13 shows measured thrusts for oscillating pad- dling experiments with various oscillation amplitudes. As shown in the figure, thrusts in x-direction have asym- metric magnitudes, whereas thrusts in y-direction follow a symmetric way. Based on this result, the capability of generating vectored trusts of the ePaddle module in the oscillating mode is experimentally confirmed as well. Fig. 15 Rolling of an ePaddle module on even ground Generating vectored thrusts in aquatic environment The oscillation period is 1.5 s and the paddle-shaft is placed at its outmost position along the x-axis a b 0 30 60 90 120 150 180 0 5 10 15 Drawbar pull [N] Angular position of the wheel [deg] 0 30 60 90 120 150 180 -50 -40 -30 -20 -10 0 Vertical force [N] Angular position of the wheel [deg] Fixed lugged wheel ls s s s = 10 mm l = 14 mm l = 18 mm l = 22 mm a b 0 30 60 90 120 150 180 0 5 10 15 Drawbar pull [N] Angular position of the wheel [deg] 0 30 60 90 120 150 180 -50 -40 -30 -20 -10 0 Vertical force [N] Angular position of the wheel [deg] Fixed lugged wheel ls s s s = 10 mm l = 14 mm l = 18 mm l = 22 mm Fig. 14 Soil-ePaddle interaction mechanics. Comparison of a draw- bar pulls and b vertical forces generated by a fixed lugged wheel (lug height = 13 mm) and by the ePaddle mechanism at various of sinkage lengths and 60° inclination angle -0.1 -0.05 0.05 0.1 0.15 Force in x-direction (N) 0 0.5 1 1.5 -0.2 -0.1 0 0.1 0.2 0.3 Time (s) Force in y-direction (N) a b 0 20deg 25deg 30deg 35deg 40deg 45deg 50deg Fig. 13 Thrusts generated in oscillating paddling mode. Measured thrusts in a x-direction and in b y-direction with different oscillation amplitudes The oscillation period is 1 5 s and the paddle-shaft is -0.1 -0.05 0.05 0.1 0.15 Force in x-direction (N) 0 0.5 1 1.5 -0.2 -0.1 0 0.1 0.2 0.3 Time (s) Force in y-direction (N) a b 0 20deg 25deg 30deg 35deg 40deg 45deg 50deg Fig. 13 Thrusts generated in oscillating paddling mode. Measured thrusts in a x-direction and in b y-direction with different oscillation amplitudes. The oscillation period is 1.5 s and the paddle-shaft is placed at its outmost position along the x-axis a 0 30 60 90 120 150 180 0 5 10 15 Drawbar pull [N] Angular position of the wheel [deg] Fixed lugged wheel ls s s s = 10 mm l = 14 mm l = 18 mm l = 22 mm b 0 30 60 90 120 150 180 -50 -40 -30 -20 -10 0 Vertical force [N] Angular position of the wheel [deg] Fig. Generating vectored thrusts in aquatic environment Figure 12 shows the experimental results of the rota- tional paddling modes with the paddle-shaft locating at an eccentric position along the y-axis. Several distances between the paddle-shaft and the wheel center were used in the experiments.i Experimental results in Fig. 12 confirm that asymmet- ric thrusts can be generated in the x-direction by placing Force in x-direction [N] Force in y-direction [N] a b Fig. 12 Thrusts generated in rotational paddling mode. Measured thrusts in a x-direction and in b y-direction with eccentric distances between the paddle-shaft and the wheel center varying from 0 to 50 mm and the wheel rotates at 50 rpm along the direction. The number on the line indicates the eccentric distance in millimeter (mm) Force in x-direction [N] Force in y-direction [N] a b Fig. 11 Sandy testbed for measuring soil-ePaddle reaction mechan- ics. a Assembled testbed. b Schematic drawing of the data acquisi- tion system Angular position of the ball-bearing screw and angular positions of the three joints of the ePaddle module were, respectively, measured by an incremental encoder (E6A2- CWZ3E, OMRON; Japan) and three absolute encoders (RE22, RLS, Slovenia), both of which were sampled at 166 Hz through a digital input/output board (NI-6001, National Instrument; USA) on a PC running a Windows XP operating system. The force signals were sampled by Table 2 Soil parameters Parameters Unit Value Cohesion stress c Pa 400 Soil friction angle φ deg 38.1 Soil specific weight γ kg/m3 1480 Adhesion stress ca Pa 66 Lug–soil friction angle δ deg 10.4 Fig. 12 Thrusts generated in rotational paddling mode. Measured thrusts in a x-direction and in b y-direction with eccentric distances between the paddle-shaft and the wheel center varying from 0 to 50 mm and the wheel rotates at 50 rpm along the direction. The number on the line indicates the eccentric distance in millimeter (mm) Sun et al. Robot. Biomim. (2016) 3:8 Page 8 of 11 the paddle-shaft at an eccentric position with respect to wheel center along the y-direction. It implies that vec- -0.1 -0.05 0.05 0.1 0.15 Force in x-direction (N) 0 0.5 1 1.5 -0.2 -0.1 0 0.1 0.2 0.3 Time (s) Force in y-direction (N) a b 0 20deg 25deg 30deg 35deg 40deg 45deg 50deg Fig. 13 Thrusts generated in oscillating paddling mode. Measured thrusts in a x-direction and in b y-direction with different oscillation amplitudes. Obstacle‑negotiating capability l l f h l d Results in Fig. 14 reveal the following advantages of the ePaddle module rolling on sandy terrain. The ePaddle mechanism is able to insert the paddle into the soil ear- lier in the cycle and depart from the soil later to increase the drawbar pull and vertical force over a wider range. Compared with the fixed lugged wheel with a maximum sinkage length of 18 mm, the maximum drawbar pull of the ePaddle module at 18 mm sinkage length is increased by 53 %. In addition, the ePaddle mechanism is able to significantly enhance the vertical reaction force on sandy soil. Experimental results of wheeled rolling on even ground, as shown in Fig. 15, confirmed that by locating the pad- dle-shaft at a proper position, the paddle-ground colli- sion can be completely avoided. Wood blocks of 100 mm height (which is about 1.78 times of wheel radius) were placed on the ground to test the step-climbing capability of the ePaddle module. Experimental results, as shown in Fig. 16, verified that the ePaddle mechanism has great advantage on climbing high obstacle thanks to the paddle’s motion. Sun et al. Robot. Biomim. (2016) 3:8 Page 9 of 11 Fig. 16 Results of step-climbing experiment. An ePaddle module successfully climbed a 100 mm height wood block Fig. 16 Results of step-climbing experiment. An ePaddle module successfully climbed a 100 mm height wood block Fig. 19 Experiment result of the paddle-aided stair climbing. The paddle-shaft is placed at its upmost position inside the wheel. Pad- dle’s motion enables the robot to climb the step Fig. 16 Results of step-climbing experiment. An ePaddle module successfully climbed a 100 mm height wood block Fig. 19 Experiment result of the paddle-aided stair climbing. The paddle-shaft is placed at its upmost position inside the wheel. Pad- dle’s motion enables the robot to climb the step Fig. 17 Results of the climbing experiments on a slope of 45° ePaddle module has great obstacle-negotiating capability when accessing rugged obstacles.h The prototype robot, as shown in Fig. 5, was used to test the step-climbing capability of an ePaddle-based quadruped robot. Results shown in Fig. 19 confirmed that the robot is able to climb the stair due to the paddle– stair interaction. This result confirmed that the paddle’s motion is useful during stair climbing, even the stair is slippy to wheels. Fig. 17 Results of the climbing experiments on a slope of 45° References 1. Murphy RR. An overview of robots from USA at Fukushima and the tsunami recovery. In: Special forum report at 2011 IEEE international conference on robotics and automation (ICRA’11). Shanghai, China 2011 1. Murphy RR. An overview of robots from USA at Fukushima and the tsunami recovery. 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Conclusions In this paper, prototype designs of an ePaddle mecha- nism and an ePaddle-based quadruped robot have been proposed. By integrating paddles into a traditional wheel mechanism, the ePaddle mechanism is superior to other locomotion mechanisms due to its advantage in locomo- tion versatility and terrain adaptability. Fig. 18 The ePaddle module is climbing stacks of wood poles Principles of multi-terrain locomotion modes of the ePaddle mechanism have been discussed and experimen- tally verified with the prototypes on fabricated testbeds.i i Experimental results have confirmed that paddle’s motion, which is actively controlled by locating the pad- dle-shaft, is useful in all the feasible locomotion modes. For example, paddles can generate vectored thrusts in aquatic environments with two paddling modes. On sandy terrains, paddle’s motion can generate larger draw- bar pull and vertical forces than traditional fixed-lug wheel. In the wheeled rolling mode, paddles can help the robot to climb high obstacles. Fig. 18 The ePaddle module is climbing stacks of wood poles Experimental results, as shown in Fig. 17, confirmed that the ePaddle module is able to climb a slope of up to 45°. In our future study, coordinating movements among the four ePaddle modules in the prototype robot will be con- sidered to further improve the mobility on rough terrains. Optimization of the locomotion modes taking the ener- getic efficiency into account will be investigated as well. Mobility on rough terrains is tested with stacks of wood poles with heights of 210 and 330 mm as shown in Fig. 18. The paddle can easily firmly hook on the poles due to their rugged shape, which suggested that the Page 10 of 11 Page 10 of 11 Sun et al. Robot. Biomim. (2016) 3:8 the 2009 IEEE/RSJ international conference on intelligent robotics and systems (IROS’09), 2009, pp. 402–403. St. Louis, USA the 2009 IEEE/RSJ international conference on intelligent robotics and systems (IROS’09), 2009, pp. 402–403. St. Louis, USA Author details 1 1 Department of Robotics, Ristumeikan University, 1‑1‑1 Noji Higashi, Kusatsu, Shiga 525‑8577, Japan. 2 Department of Mechanical and Aerospace Systems Engineering, Tokyo Institute of Technology, Ookayama, Megoru‑ku, Tokyo 152‑8552, Japan. 3 School of Mechatronic Engineering and Automation, Shanghai University, 149 Yanchang Road, Shanghai, China. 16. Shores B, Minor M. Design, kinematic analysis, and quasi-steady control of a morphic rolling disk biped climbing robot. In: Proceedings of the 2005 IEEE international conference on robotics and automation (ICRA’05), 2005, pp. 2721–2726. Barcelona, Spain Acknowlegements 17. Shen SY, Li CH, Cheng CC, Lu JC, Wang SF, Lin PC. Design of a leg-wheel hybrid mobile platform. In: Proceedings of the 2009 IEEE/RSJ interna- tional conference on intelligent robots and systems (IROS’09), 2009, pp. 4682–4687. St. Louis, USA This study was supported by “Strategic Research Foundation Grant-aided Project for Private Universities (2013–2017)” from Ministry of Education, Cul- ture, Sports, Science and Technology, Japan. The fabrication assistance from Material and Manufacturing Center of Ritsumeikan University is also gratefully acknowledged. 18. Huang KJ, Chen SC, Chou YC, Shen SY, Li CH, Lin PC. Experimental valida- tion of a leg-wheel hybrid mobile robot Quattroped. In: Proceedings of the 2011 IEEE international conference on robotics and automation (ICRA’11), 2011, pp. 2976–2977. Shanghai, China Authors’ contributions YS proposed the ePaddle mechanism and locomotion modes, developed the controller network, conducted terrestrial locomotion experiments, and wrote the manuscript. YY developed the prototypes, conducted soft-terrain experi- ments, analyzed the data, and wrote the manuscript. SM designed the study and developed the methodology. HP conducted the aquatic experiments, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript. y pp 13. Okada T, Botelho WT, Shimizu T. Motion analysis with experimental verifi- cation of the hybrid robot PEOPLER-II for reversible switch between walk and roll on demand. Int J Rob Res. 2010;29(9):1199–221. 14. Steffan E, Das T. Locomotion of circular robots with diametrically translat- ing legs. In: Proceedings of the ASME 2009 dynamic systems and control conference (DSCC’09), 2009, pp. 1–7. Hollywood, CA, USA 15. Yan J, Agrawal S. Rimless wheel with radially expanding spokes: dynam- ics, impact, and stable gait. In: Proceedings of the 2004 IEEE international conference on robotics and automation (ICRA’04), 2004, vol 4, pp. 3240–3244. New Orleans, LA, USA Competing interests 19. Consi T, Bingham S, Chepp J, Erdmann T, Mehrotra A, Ringstad J, Zhao B. Amphibious robots as rapidly deployable near-shore observatories. In: Proceedings of the OCEANS 2010, pp. 1–6. Washington DC, USA The authors declare that they have no competing interests. Received: 1 March 2016 Accepted: 10 May 2016 Received: 1 March 2016 Accepted: 10 May 2016 20. Aponick T, Bernstein C. Countermine operations in very shallow water and surf zone: the role of bottom crawlers. In: Proceedings of the OCEANS 2003, pp. 1931–1940. San Diego, CA, USA 21. Low K, Zhou C, Ong T, Yu J. Modular design and initial gait study of an amphibian robotic turtle. In: Proceedings of the 2007 IEEE international conference on robotics and biomimetics (ROBIO’07), 2007, pp. 535–540. Sanya, China 36. Pu H, Sun Y, Ma S, Gong Z. Experimental study on the oscillating paddling gait of an epaddle mechanism with flexible configuration. Adv Robot. 2014;28(11):769–80. 39. Ma S, Tomiyama T, Wada H. Omni-directional static walking of a quadru- ped robot. IEEE Trans Robot. 2005;21(2):152–61. 38. Pu H, Sun Y, Ma S, Gong Z. Experimental study on oscillating paddling gait of an eccentric paddle mechanism. In: Proceedings of the 2012 IEEE international conference on robotics and biomimetics (ROBIO’12), 2012, pp. 187–192. Guanzhou, China (Best Conference Paper Award Finalist). 37. Pu H, Sun Y, Yang Y, Ma S. Modeling of the oscillating-paddling gait for an epaddle locomotion mechanism. In: Proceedings of the 2013 IEEE international conference on robotics and automation (ICRA’13), 2013, pp. 3414–3420. Karlsruhe, German 36. Pu H, Sun Y, Ma S, Gong Z. Experimental study on the oscillating paddling gait of an epaddle mechanism with flexible configuration. Adv Robot. 2014;28(11):769–80. 37. Pu H, Sun Y, Yang Y, Ma S. Modeling of the oscillating-paddling gait for an epaddle locomotion mechanism. In: Proceedings of the 2013 IEEE international conference on robotics and automation (ICRA’13), 2013, pp. 3414–3420. Karlsruhe, German 38. Pu H, Sun Y, Ma S, Gong Z. Experimental study on oscillating paddling gait of an eccentric paddle mechanism. In: Proceedings of the 2012 IEEE international conference on robotics and biomimetics (ROBIO’12), 2012, pp. 187–192. Guanzhou, China (Best Conference Paper Award Finalist). 39. Ma S, Tomiyama T, Wada H. Omni-directional static walking of a quadru- ped robot. IEEE Trans Robot. 2005;21(2):152–61. 32. Pu H, Zhao J, Sun Y, Ma S, Luo J, Gong Z. Non-reciprocating legged gait for robot with epicyclic-gear-based eccentric paddle mechanism. Robot Auton Syst. 2015;68:36–46. doi:10.1016/j.robot.2015.02.004. http://www. sciencedirect.com/science/article/pii/S09218890150%00226. 33. Yang Y, Sun Y, Pu H. Paddle trajectory generation for accessing soft terrain by an ePaddle locomotion mechanism. 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https://openalex.org/W3081196916	http://www.acrn-journals.eu/resources/jofrp09k.pdf	English	null	Transition Finance and Markets	ACRN journal of finance and risk perspectives	2,020	cc-by-sa	8,163	* Corresponding author. E-Mail address: Deborah.Cotton@uts.edu.au ORCID: 0000-0003-1605-6749 ABSTRACT Article history: Received 17 May 2020 Revised 29 June 2020 Accepted 01 July 2020 Published 20 August 2020 The increased focus and agreement on the requirement for the planet to be more sustainable has led to an array of new research and financial products. The new buzz phrase is transition financing which is being seen as the path to achieving a sustainable world. The Development Assistance Committee (DAC) in the Organisation for Economic Co-operation and Development (OECD, 2019) has the main objective of transition finance is to optimise access to finance for sustainable development to avoid financing gaps or socio-economic setbacks. This chapter examines some of the products and markets in current use by financial institutions and investors. It describes their use and recent research in this area as well as some gaps in this research. Keywords: Transition finance Green bonds Environmental markets https://doi.org/10.35944/jofrp.2020.9.1.011 ISSN 2305-7394 Transition Finance and Markets Deborah Cotton* UTS Business School, University of Technology Sydney ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 (i) Bonds This issue is shown in a quote from one sustainable finance head who said “We need investors to support bonds and other financial tools that help a transition – be those called transition bonds, or dark green (or brown) bonds, rather than just looking for pure green”. In fact we find that the EU is starting to move in this direction with their taxonomy including ‘transition’ into its lexicon when discussing green finance. Many working in this sector suggest that there is not enough involvement from the institutional investors. Avery (2019) quotes Rhian-Mari Thomas of the Green Finance Institute that “The perceived risk in emerging markets – FX risk, political risk, a lack of secondary market and liquidity – means the large amounts of capital are not getting to where they need to go. We need more investable channels and tools; possibly aggregating different projects, using warehousing techniques, for example, as well as involving concessional and philanthropic capital to credit enhance or reduce risk”. Ehlers and Packer (2017) found an increase in the financing of investments through green bonds and conclude that they should be the easiest form of finance for green projects. However, they also found that the variation in the definition of green bonds was significant. Wisniewski and Zielinski (2019) also indicated the need for clear guidelines on the definition and authorisation of green bonds and the scarcity of research in this area. So, while we find a push for a clear taxonomy and clarification there remain instruments such as unlabelled climate aligned bonds being issued. Their paper, and one by Hachenberg and Schiereck (2018), question if they are selling at a premium and are therefore not achieving are achieving an attractive risk-return relationship. There is some evidence that they trade tighter when compared to non-green bonds and government related bonds, in particular for A rated bonds. They suggest the higher price of the green bonds may be due in part to the additional certification and verification required. Zerbib (2019) found an average two basis point yield for green bonds lower than conventional bonds however demand remained strong for these environmental bonds in their study of European and US bond markets. Sartzetakis (2019) looks specifically at green bonds as the preferred instrument to use for financing the transition to a low carbon economy. However, there are a few quite specific and interesting issues which are discussed. (i) Bonds The greatest level of growth and innovation in recent years is in the use of bonds as a financing instrument. Takatsuki and Foll (2019) (AXA investment managers in London) wrote guidelines for transition bonds which were published by AXA in June 2019. They suggest there hasn’t been the growth in this market into what they refer to as the bulk of the real world economy i.e. heavy industry and manufacturing. It has been limited to “supranational, sovereign and agency (SSA) issuers, commercial banks and electricity utilities in Europe and North America”. The DAC of the OECD investigated the support and linkages between sources of finance, both public and private. They find that domestic resources are the largest source of funding for the majority of countries studied. It was also found that they are highly dependent on public external support in the early stages of the transition. The International Energy Agency’s (IEA) Sustainable Development scenario requires demand for coal to fall by more than 50% by 2040 to achieve the 1.50 IPPCC goal. This impacts heavily on the electricity generation sector which currently is at around 40% from fossil fuels. These bonds will focus on greenhouse gas intensive industries who do not have access to green finance but will require significant financing to make the changes required. Access to date for green bonds has been focussed on the use of the proceeds on climate and environmental activities such as wind farms and solar panels. Broadly the activities for which these transition bonds could finance include; • Cogeneration plants • Carbon capture storage • Waste to energy • Gas powered ships • Aircraft alternative fuels https://doi.org/10.35944/jofrp.2020.9.1.011 ISSN 2305-7394 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 Increase use of recycled materials, smelting reduction and other energy reducing methods for the targeted industries Increase use of recycled materials, smelting reduction and other energy reducing methods for the targeted industries Avery (2019) suggests that while many finance organisations want to change their lending practices they are essentially caught in the clients’ needs. A greater collaboration of these organisations with their clients during the structuring and deal making stages is needed. Further they suggest the green bonds are failing to change these practices or their clients’ plans for investment and that sustainability linked loans are a better prospect. (i) Bonds One of these is the question of intergenerational equity where he asks whether the current generation should bear all the costs of climate change mitigation. While this appears a more of a philosophical question, he addresses it by suggesting debt is an ideal tool for spreading the cost across generations. Specifically, a combination of taxes and debt was recommended. This appears a good method but does this address the social optimum and the aging population including the many issues around this such as the changes that will occur due to climate change? Here we might consider the significant changes to the types of work, where and how we work as well as the health aspects that will be affected. Are financial instruments used going to have an impact on any, or all, of these issues plus the many more that could be suggested? Beyond this Sartzetakis (2019) looks at the amounts of funding that are going to be required to renew and change the infrastructure to meet the new energy source needs for the low carbon economy. A real issue that is already evident in the green bond market is the concern around greenwashing and transparency. The Green Bonds State of the Market 2018 (Climate Bonds Initiative, 2019) report provided the following information on the green bond market in 2018 in US Dollars: Table 1. Top 3 Countries and their Top Financiers of Green Bonds in 2018 Country $USD Issuer $USD USA 34.2bn Fannie Mae 20.1bn China 31bn Industrial Bank 9.6bn France 14,2bn Republic of France 6bn Source: Green Bonds State of the Market 2018 Table 1. Top 3 Countries and their Top Financiers of Green Bonds in 2018 Source: Green Bonds State of the Market 2018 Source: Green Bonds State of the Market 2018 We see the dominance of sovereign funds and large financial organisations. Importantly though the development banks which were early adopters of this type of investment. Geographically the largest proportion is in the US, closely 138 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 followed by other developed economies such as Canada and Germany. Interestingly there was significant growth in China but this level of growth had all but disappeared by 2017. g pp y The growth has been strong in recent years and appears to be stabilising. (i) Bonds While the majority of the funding is still into renewable energy there has been an increase into low carbon buildings and energy efficiency. Other areas of growth include transport while waste, land use and adaptation remain small. g p p A better understanding of the market is needed by investors for this market to grow for it to have the desired effects. This would include information of each project and its’ progress including financial returns and environmental benefits. It is only through clarity of these that the green bond market will be a useful instrument that is confidently invested in by the large fund managers. This concept, and the greenwashing mentioned earlier, engenders an interest in understanding more around the taxonomy and the different metrics used. For example, we can consider the UN’s Sustainable Development Goals (SDGs), of which there are 17, or the EU taxonomy for sustainable activities, which is applied to screening criteria for 67 activities, or the Task Force on Climate-related Financial Disclosures (TCFDs) which focusses on climate issues more specifically than the other two. Future research topics: • Easy research includes the need for some updating from previous work due to the relative newness of, and growth in, green bonds in recent years – such as who are the investors, is public support still required, what is the growth and in what types of investments e.g. wind farms or waste? • What would/could transition bonds look like? If investing in e.g. waste to energy or smelting reduction technologies would these instruments be positioned differently or structured differently? Is there a market for these if institutional investors are still reticent about the more familiar green energy bonds? • What would/could transition bonds look like? If investing in e.g. waste to energy or smelting reduction technologies would these instruments be positioned differently or structured differently? Is there a market for these if institutional investors are still reticent about the more familiar green energy bonds? • Some recent research suggests that green bonds are expensive compared to their non-green bond counterparts but it is not clear why? Is it really the case or are the risks not being properly accounted for? Does the uncertainty of what green bonds really are add to these issues? (i) Bonds • It has been suggested that bonds are the best way to spread the cost of climate change mitigation across generations – is this the case? Should it be spread in the first place and why? • Green bond growth in China was strong but this growth has slowed. Why is this the case or in fact is it correct? This would need to be verified using up to date data and this may provide an understanding of why have they moved their focus away from these investments. (ii) Low-carbon transition Transition pathways include a broad range of risks and uncertainties, these are addressed in Narratives of Low-Carbon Transitions – Understanding Risks and Uncertainties. While these are assessing the risks in particular for the low- carbon pathways for the energy sector transition it allows some understanding of what is required for risk assessment when using the types of transition financing being discussed in chapter 9 (van Vliet, O, 2019). Displayed below as an example is Table 2 using data from the book which looks at the different types of risk in Switzerland: Table 2: Risks to successful expansion of renewable sources of electricity in Switzerland (CH) Barriers Hydropower Rooftop PV Utility scale wind/PV Offshore wind Mediterranean CSP High Investment costs Compounded by low prices Ameliorated by FIT and fees Compounded by low prices Compounded by low prices Compounded by low prices Landscape/ Visual impact Only for large hydro Minor Unwanted Low population if far offshore Low population in desert Permitting Nature compensation done Building owner’s permission needed Challenges expected Transmission line may be challenged Transmission line may be challenged Table 2: Risks to successful expansion of renewable sources of electricity in Switzerland (CH) 139 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 Barriers Hydropower Rooftop PV Utility scale wind/PV Offshore wind Mediterranean CSP Intermittency Base load and dispatchable Diurnal, weather and seasonal Diurnal, weather and seasonal Relatively stable, better in winter Thermal storage reduces Energy independence Swiss source Swiss source, need balancing Swiss source, need balancing Contributes to diversification Contributes to diversification Source: Narratives of Low-Carbon Transitions Source: Narratives of Low-Carbon Transitions Other risks associated with transition finance to a low-carbon economy may be to the economy more broadly, in particular to carbon-intensive industries. These industries may find their asset values to be adversely affected in particular with any abrupt changes to their energy sources or allowed emission outputs. As noted by Semieniuk et. al. (2019) much climate policy requires rapid support for low carbon technologies via the transition finance which may leave the carbon intensive firms with stranded assets and an inability to change fast enough. These impacts will not be isolated to a few companies but rather have a broader impact on the economy. An investigation by Owen et. al. (ii) Low-carbon transition This would be an interesting place to do this kind of analysis in particular if it could include the other costs of implementing these types of renewable energy supplies. • While there has been some research on stranded assets there remains a lack of clear understanding on how these impact not just on the companies who have them but also the broader community. Are the costs and risks of stranded assets being included in transition investing and if so how and to what extent? • While there has been some research on stranded assets there remains a lack of clear understanding on how these impact not just on the companies who have them but also the broader community. Are the costs and risks of stranded assets being included in transition investing and if so how and to what extent? (ii) Low-carbon transition (2018) found the need for a ‘finance ecosystem’ approach to ensure a range of forms of finance for low carbon investment that would support the sector. This ecosystem would include both private and public sector funding. The finance gap, or lack of funding into this market, would be a significant constraint for businesses trying to work in this low carbon sector and is most commonly found in low income countries and for longer term investment requirements. This paper also discusses the need for scaling up this funding which is currently focussed often on pilot projects – of interest for future research might be where is this funding come from? What does it look like? Is this need similar to any previous financing needs such as information technology? They also note “a lack of evaluations looking at additionality and the demonstrator effect, in order to effectively assess whether public sector support is crowding out or crowding in investment. This requires the development of assessment methodologies that address displacement of private investment and the counterfactual questions such as: how much co-financing from the private sector would have occurred without public sector support. This evidence of the impact of public sector funded interventions is needed — alongside a greater focus evaluating the green innovation impact on the wider economy — to identify where public funding can have the biggest impact on the transition to a low carbon economy.” Future Research Topics: • Barriers to entry and risks associated with this kind of investing is highlighted in the Swiss report e.g. the intermittent supply and costs of storage or impact on landscape. Some studies in different contexts have used logit analysis to determine willingness to pay to keep wind farms etc. out of particularly beautiful areas. This would be an interesting place to do this kind of analysis in particular if it could include the other costs of implementing these types of renewable energy supplies. • Barriers to entry and risks associated with this kind of investing is highlighted in the Swiss report e.g. the intermittent supply and costs of storage or impact on landscape. Some studies in different contexts have used logit analysis to determine willingness to pay to keep wind farms etc. out of particularly beautiful areas. Systems and Policies and the New Green Deal Discussion around what kind of policy is required to achieve the emission targets set in Paris in 2016 has occurred since then. The difference in the most recent discussions is less about who needs to agree and to what levels and more about what needs to happen in the finance sector to facilitate the changes required. There has been a certain amount of agreement that a policy mix is required which would include the use of “fiscal instruments, targets and standards; public private co-funding schemes; financial regulation; and disclosure practices.” (Lamperti et. al., 2019, p1.) The focus in their paper is not on simply scaling up the existing financial sources but more on the quality and de-risking these investments. They found a significant difference in what types of research and development was being funded by public and private i.e. public funding focussed on less commercial ventures such as tidal and wave energy while private funding was predominantly in the established technologies such as wind. Further the private funds only 140 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 entered the higher risk areas once there was existing public funds invested via subordinated debt. In their final policy insights they state that “Institutional investment may not be enough to vastly increase renewable energy investments to a scale consistent with mitigation scenarios.” (Lamperti et. al., 2019, p. 11). Additionally, they call for investors to be patient, not overly risk averse and be prepared to take on projects that will not provide strong returns. As discussed earlier the SDG’s are taking their place in identifying the areas of interest or application of investments. There has been some research to date on the SDG’s with a few explaining what they do and their role in the growth of sustainable development (Wood et. al. (2018), Leal Filho et. al. (2018), Schmidt-Traub et. al. (2017)). The need for these to be given a greater level of priority in research for interdisciplinary research, the development of local level research and to highlight the scientific results to a greater range of stakeholders were considered an important use of these goals. Environmental goals were considered to have a large focus among the SDGs and that the interaction among these had significant benefits as seen in Wood et. al. (2018). Systems and Policies and the New Green Deal The EU Taxonomy is the most recent of the metrics and works alongside the SDGs, it was devised by the European Commission to inform its work on the action plan for financing sustainable growth which was established in 2018. It sets out criteria for 67 activities that are making a contribution to climate change mitigation and works with the mandate of the technical expert group on sustainable finance. In brief the report states that “The EU Taxonomy is a tool to help investors, companies, issuers and project promoters navigate the transition to a low-carbon, resilient and resource-efficient economy.” (EU Technical Expert Group on Sustainable Finance, p. 2). Not surprisingly there has as yet been little research on this taxonomy however we do find a short comment by Lai (2019) where he states that Japanese asset managers are sceptical of the rule based taxonomy and their preference is for a more market led approach. Schmidt-Traub et. al. (2017) investigated the gaps in scope and coverage of the various indexes and dashboards which is of interest to future researchers in evaluating the various metrics that are currently in use. Other papers explore more specific applications including in nursing, victims of unsustainability and wicked problems such as the challenge of food safety versus food security. There are many more specific areas to look at which could be better understood by reading some of the existing work by authors such as Rush (2019), Benton and Shaffer (2016) and Kopnina (2015). The TCFDs were recommended in 2017 and are strictly focused on climate related issues and are more similar to the EU Taxonomy than the broader SDGs. They provide recommendations on how to disclose any climate related financial risks as well as opportunities. It provides recommendations around governance issues such as board oversight of these issues as well as management’s role in assessing and managing these risks and opportunities. Strategy and risk management practices are recommended as well as guidance around metrics disclosure, scopes (1, 2 and 3) as well as targets used to manage the risks. To date much of the research around TCFD’s has been predominantly in the form of commentaries such as Tyagi (2018) who stated that the oil and gas industry are possibly the only sector with the necessary expertise and position to adapt and strengthen the risks and opportunities the TCFDs are designed to identify. Systems and Policies and the New Green Deal Herman (2105) continued this discussion into the impact on equality finding the concepts of this earlier version to in fact have a furthering of the unequal distribution on environmental and economic assets. Four years later White (2019) offered some notes on how some of the disengagement shown in the vote in the US Congress in 2019 could be ameliorated. He states that “This paper is written from the sense that it may well be more productive now to acknowledge that all conceivable programs for just transition are going to be socio-technical in nature, multi-scalar and, by definition, concerned with designing low-carbon futures.” (p. 1). h di i f h i l k i i fi ll l h h b k d i , , y , g g (p ) The discussion of the progress requires a look at transition finance on a smaller scale – what are the banks doing with personal lending and small business loans? This is the less ‘sexy’ side of transitioning finance but may be interesting to better understand and may provide some interesting research opportunities. We know that many of the large financial institutions are offering cheaper loans if you are putting solar panels on your home or installing a water tank. Similarly small businesses have similar ‘energy saving’ savings on loans available as well as the more direct interest rate reductions if they are in the business of green technologies. In Australia a comparison website (Finder, 2019) state “A green personal loan is a personal loan specifically designed to fund the purchase of specific energy- efficient products, such as electric or hybrid cars, energy-efficient air conditioning or solar panels. These loans can be secured or unsecured and have terms ranging from six months up to seven years. They also tend to feature flexible borrowing limits based on the items you want to purchase, and many lenders allow you to make early repayments without penalty. Green personal loans are a good choice for most borrowers who have a good credit score.” So while not exactly indicating much of a discount we find globally this is more clear. The Canadian bank RBC offer a 1% discount on their interest rates for loans over $5,000 when purchasing certain energy saving products. Systems and Policies and the New Green Deal Table 3: % of GDP in Green Investment by Country Source: Barbier (2010) A little later Schwartzman (2011) looked at the deal from the perspective of the success of the New Deal enacted during the Great Depression in the 1930’s. He argues that the aim of that deal was to save capitalism and that the class struggle that ensued was what resulted in the formation of the industrial unions in 1936. He goes on to suggest that this New Green Deal could provide an opportunity for another such research stating one of the goals i.e. the creation of worker owned factories to be a possible catalyst. While this angle of the research is a little different from the expected it is often these completely new ways of analysing an issue that are the most interesting and innovative/productive. Herman (2105) continued this discussion into the impact on equality finding the concepts of this earlier version to in fact have a furthering of the unequal distribution on environmental and economic assets. Four years later White (2019) offered some notes on how some of the disengagement shown in the vote in the US Congress in 2019 could be ameliorated. He states that “This paper is written from the sense that it may well be more productive now to acknowledge that all conceivable programs for just transition are going to be socio-technical in nature, multi-scalar and, by definition, concerned with designing low-carbon futures.” (p. 1). Table 3: % of GDP in Green Investment by Country Source: Barbier (2010) Table 3: % of GDP in Green Investment by Country Source: Barbier (2010) Source: Barbier (2010) Source: Barbier (2010) A little later Schwartzman (2011) looked at the deal from the perspective of the success of the New Deal enacted during the Great Depression in the 1930’s. He argues that the aim of that deal was to save capitalism and that the class struggle that ensued was what resulted in the formation of the industrial unions in 1936. He goes on to suggest that this New Green Deal could provide an opportunity for another such research stating one of the goals i.e. the creation of worker owned factories to be a possible catalyst. While this angle of the research is a little different from the expected it is often these completely new ways of analysing an issue that are the most interesting and innovative/productive. Systems and Policies and the New Green Deal In a similar vein Eccles and Krzus (2019) conducted a field experiment prior to the publication of the TCFDs on fifteen of the largest oil and gas companies by market capitalisation to determine how easily they could be implemented. They found that a number of the required disclosures were already in existence as voluntary and recommended further studies be conducted to see how these have been implemented since the publication. The Green New Deal is not a new idea as such having been first termed by Thomas Friedman in 2007 to raise the idea of a transition away from fossil fuels. It appeared to be gaining new ground in recent years with a resolution going to US Congress in 2019 however it was voted down in the Senate by 57 to 0. So while it is not looking likely in the current political environment in the US it appears to have greater resonance in the banking and investment community who are looking more into sustainable products for the long term. The goals of this Green New Deal included job creation in the transition in areas such as sustainable agriculture, conservation and infrastructure; workers displaced from their current jobs to receive income and benefits as they transition to alternative work; public, community and worker ownership of the energy system; and end all subsidies for fossil fuels and impose a greenhouse gas fee or tax. g The previous version of this green new deal had a large amount of academic research undertaken looking at it from a variety of perspectives. An opinion piece in Nature by Barbier (2010) gave two key reasons for countries to get behind this new transition; “First, international competitiveness: those countries that have spent large amounts on green projects are now better placed in the race for technological superiority. Second, deficit reduction: reducing dependency on foreign oil and raising cash through carbon taxes or auctioned permits will ease national debts.” He discusses leaders and laggards and cites worsening effects of climate change including energy insecurity, environmental degradation and global poverty as some of the results on inaction. Table 3 below shows data from 141 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 2009 as described by Barbier (2010) showing the percentage of GDP in green investments by country compared to the 1% target of the deal. the 1% target of the deal. Systems and Policies and the New Green Deal • What changes have or are likely to occur under the Green New deal and how likely is the creation of worker owned factories being created? • There is a great deal to explore in how or if the Green New Deal is implemented and a vast array of consequences under any guise – all of which encourage interesting research exploration e.g. who are the leaders and the laggards and why? • There is a great deal to explore in how or if the Green New Deal is implemented and a vast array of consequences under any guise – all of which encourage interesting research exploration e.g. who are the leaders and the laggards and why? gg y • A little researched area of finance in general are the lending practices of the large financial institutions around the world which would be greatly enhanced with an investigation of the lending practices associated with the view to essentially green lending on a small and medium scale. Simpson et. al. (2016) looked at the GFC as a driver for more responsible mortgage lending and may provide a sound way to undertake this kind of analysis. Systems and Policies and the New Green Deal The World GBC Europe Regional Networks (2018) wrote the report ‘Creating an Energy Efficient Mortgage for Europe: Towards a New Market Standard’ which sets out a plan for how mortgage lenders and borrowers can help tackle climate change by improving millions of properties across Europe. It proposes energy efficiency mortgages to be made available across Europe and calls on lenders, industry and government to work together to achieve these goals. There appears to be a growth area for transition finance in the banking sector but little to no growth in the research covering this new and interesting part of the ‘road to a sustainable planet’. 142 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 Future Research Topics: • The need for investors to be patient and willing to accept a lower return is the opposite to what is mostly in place in fund management and appears contradictory. So what does this mean these investments look like? A h ddi i i i fi i l i i hi l l i d? • The need for investors to be patient and willing to accept a lower return is the opposite to what is mostly in place in fund management and appears contradictory. So what does this mean these investments look like? Are they a mere addition to existing financial instruments or is something completely new required? place in fund management and appears contradictory. So what does this mean these investments look like? Are they a mere addition to existing financial instruments or is something completely new required? p g pp y Are they a mere addition to existing financial instruments or is something completely new required? ey a mere addition to existing financial instruments or is something completely new required? • While the SDG’s are still very new there is an array of research opportunities including how or if they can be applied globally or would they have to be managed to fit within other economies? • Does the new taxonomy emerging help investors to better understand the green bonds and other transition finance and their investment prospects? • The work done by Eccles and Krzus (2019) prior to the publication of the TCFDs would be interesting to follow up and determine exactly how well they are managing post publication. Provinces and States Cities Source: ICAP, Status Report 2020 Source: ICAP, Status Report 2020 The best known and most widely traded is the EU ETS and has had the most research already undertaken on it. Other schemes and research undertaken have come and gone including the Australian Emissions Trading Scheme where Australia was an early adopter however the political environment caused its demise. Interestingly this political effect has been investigated recently in Britain, Germany, Australia and New Zealand with a greater level of investigation required to be able to form a comprehensive view of the impact. A few reviews have been undertaken of emissions trading (Ranson et. al. (2016), Ellerman et. al. (2015), Jotzo et.al (2018)) which provide some interesting issues which may provide further investigation. The importance of linkages between schemes and across jurisdictions which may provide a better global coverage and accountability is discussed both explicitly in some of these and other papers. As noted in Table 4 above, the city-based approach in China has dominated but as Jotzo et.al. (2018) show there is a broader approach being applied, in this growing part of the global economy a better understanding of what is taking place there would benefit greatly the growth in knowledge of global emissions management. Other research has investigated the carbon emissions schemes and corporate responsibility (Kolk and Pinkse, 2017). g p p y ( , ) The lesser known markets include water quality (and quantity) markets, habitat and biodiversity markets and wetland markets in the US. Below is a summary of these markets: Table 5: U.S. Environmental Markets Market Summary Water Quality Goal to reduce pollution of waterway by land managers to meet the Clean Water Act requirements. To date it has not been widely adopted. At trading’s core, a buyer (e.g. a pollution source such as a municipal wastewater facility) purchases water quality improvements, or credits, from a seller (e.g. a farmer installing a buffer along a stream to capture sediment runoff) that reduces its pollutants beyond what it is required to do. In order for trades to occur, the seller needs to reduce pollution more cost effectively than the buyer. Both buyers and sellers will need to control their own pollution to some minimum level, or baseline, before generating credits (U.S.EPA, 2007, p29). Source: United States Department of Agriculture Markets as transition instruments Globally there are a vast array of environmental markets. The major markets we think of are those related to climate change/energy/carbon such as the European Emissions Trading Scheme and the California Emissions Trading Scheme. We will commence by reviewing these markets and then proceed to investigate the other less researched markets. The most recent report by the International Carbon Action Partnership (ICAP) provides information on these markets including how they are traded, sectoral coverage and allowance prices. It suggests that almost 1 in 6 of the global population lives in a country/region or city in which an ETS is in force. Currently the operating ETS are set up in Table 4 below in order of levels of coverage of a scheme: Table 4: Emissions Trading Schemes Supranational Countries Provinces and States Cities EU member states + Iceland, Lichtenstein and Norway Kazakhstan California Beijing Mexico Connecticut Chongqing New Zealand Delaware Saitama Republic of Korea Fujian Shanghai Switzerland Guangdong Shenzhen Hubei Tianjin Maine Tokyo Maryland Massachusetts New Hampshire Table 4: Emissions Trading Schemes 143 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 Supranational Countries Provinces and States Cities New Jersey New York Nova Scotia Québec Rhode Island Vermont Source: ICAP Status Report 2020 Provinces and States Cities Water Quantity Water quantity markets, including groundwater offsets and in-stream buybacks, operate when water rights are appropriated for non-consumptive use, or when water rights are shifted between users to reallocate resources within a watershed. Wetland Markets for wetlands are used to create incentives for landowners to improve the ecosystem of their land and form part of the Clean Water Act Section 404. Biodiversity and Habitat Markets Environmental markets for habitats create incentives for landowners to improve ecosystem services on their lands. These markets are often incentivized by regulatory programs such as the Endangered Species Act, though they may also be voluntary. Carbon Markets Increasing atmospheric concentrations of greenhouse gases (GHGs) such as carbon dioxide, methane, and nitrous oxide cause changes in temperature and climate. Environmental markets for carbon and GHG can provide funding for conservation practices that decrease carbon dioxide or other GHG emissions. Source: United States Department of Agriculture Table 5: U.S. Environmental Markets Water quantity markets, including groundwater offsets and in-stream buybacks, operate when water rights are appropriated for non-consumptive use, or when water rights are shifted between users to reallocate resources within a watershed. Markets for wetlands are used to create incentives for landowners to improve the ecosystem of their land and form part of the Clean Water Act Section 404. Environmental markets for habitats create incentives for landowners to improve ecosystem services on their lands. These markets are often incentivized by regulatory programs such as the Endangered Species Act, though they may also be voluntary. Increasing atmospheric concentrations of greenhouse gases (GHGs) such as carbon dioxide, methane, and nitrous oxide cause changes in temperature and climate. Environmental markets for carbon and GHG can provide funding for conservation practices that decrease carbon dioxide or other GHG emissions. Source: United States Department of Agriculture 144 D. Cotton / ACRN Journal of Finance and Risk Perspectives 9 (2020) 137-147 When looking at social markets we don’t find the level of depth as for these more widely recognised environmental markets. However, it is clearly understood that a sustainable world includes social elements. Eizenberg and Jaboreen (2017) state that efforts toward a sustainable planet will be undermined without socially oriented practices. They suggest it requires recognition that vulnerable groups will bear a large amount of the burden of climate change for example and this is exacerbated by their lack of a voice. Provinces and States Cities Their proposition is that participation by all groups of society will increase the efficacy of any strategies towards sustainability. An important financial instrument used in this area are the social impact bonds (SIBs) and these have been widely used, and for the most part successful, in achieving their goals whether they are reducing recidivism, keeping children with their families or building affordable housing. What they are missing is a market in which they can be traded leaving them a difficult investment proposition for fund managers needing liquidity. The London Stock Exchange has listings for Charity Bonds which provides an idea of how SIBs may have an avenue for trading. Future Research Topics • Of interest to researchers looking at the broad range of environmental markets in the US would include their success rate in improving their targeted issues, the take up of these instruments and who is benefiting. We assume that the major benefactor is the environment however for these markets to be viable they require an ability to trade and financial benefits (or at least not losses) for those buying and selling in them. • Of interest to researchers looking at the broad range of environmental markets in the US would include their success rate in improving their targeted issues, the take up of these instruments and who is benefiting. We assume that the major benefactor is the environment however for these markets to be viable they require an ability to trade and financial benefits (or at least not losses) for those buying and selling in them. • There is little to no research looking at making SIBs a liquid security and if they are to achieve a greater level of importance in the implementation of social benefits as benefits to sustainability this may be a good place to start. • There is little to no research looking at making SIBs a liquid security and if they are to achieve a greater level of importance in the implementation of social benefits as benefits to sustainability this may be a good place to start. Conclusion These financial and sustainability related instruments and markets are significantly under researched using scientific and independent methodologies. There has been significant growth in these areas in recent years and therefore they have increased without much clear understanding of their impacts. 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https://openalex.org/W1997821818	https://cp.copernicus.org/articles/9/2319/2013/cp-9-2319-2013.pdf	English	null	Two ocean states during the Last Glacial Maximum	null	2,012	cc-by	13,247	Different ocean states and transient characteristics in Last Glacial Maximum simulations and implications for deglaciation X. Zhang, G. Lohmann, G. Knorr, and X. Xu Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, Bussestrasse 24, 27570 Bremerhaven, Germany Correspondence to: X. Zhang (xu.zhang@awi.de) Received: 6 July 2012 – Published in Clim. Past Discuss.: 1 August 2012 Revised: 5 July 2013 – Accepted: 13 August 2013 – Published: 15 October 2013 Abstract. The last deglaciation is one of the best con- strained global-scale climate changes documented by climate archives. Nevertheless, understanding of the underlying dy- namics is still limited, especially with respect to abrupt cli- mate shifts and associated changes in the Atlantic meridional overturning circulation (AMOC) during glacial and deglacial periods. A fundamental issue is how to obtain an appropriate climate state at the Last Glacial Maximum (LGM, 21 000 yr before present, 21 ka BP) that can be used as an initial con- dition for deglaciation. With the aid of a comprehensive cli- mate model, we found that initial ocean states play an impor- tant role on the equilibrium timescale of the simulated glacial ocean. Independent of the initialization, the climatological surface characteristics are similar and quasi-stationary, even when trends in the deep ocean are still signiﬁcant, which pro- vides an explanation for the large spread of simulated LGM ocean states among the Paleoclimate Modeling Intercom- parison Project phase 2 (PMIP2) models. Accordingly, we emphasize that caution must be taken when alleged quasi- stationary states, inferred on the basis of surface properties, are used as a reference for both model inter-comparison and data model comparison. that changes in the glacial deep ocean already occur before the last deglaciation. In combination, these ﬁndings repre- sent a new paradigm for the LGM and the last deglaciation, which challenges the conventional evaluation of glacial and deglacial AMOC changes based on an ocean state derived from 21 ka BP boundary conditions. 1 Introduction Due to its capability to redistribute large amounts of heat around the globe (e.g. Ganachaud and Wunsch, 2000), the Atlantic meridional overturning circulation (AMOC) is a key player in the global climate system. Potential changes in the operational mode of the AMOC, as a consequence of alter- ations in the hydrological cycle and greenhouse gas concen- tration, draw our concerns regarding the future fate of our climate (Meehl et al., 2007). During glacial and deglacial periods, large and abrupt changes in the climate system are thought to have repeat- edly occurred. These changes have been linked to large and abrupt shifts in the AMOC (e.g. Dansgaard et al., 1993; Bard et al., 2000; Ganopolski and Rahmstorf, 2001; Knorr and Lohmann, 2003, 2007; Liu et al., 2009; Barker et al., 2010; Menviel et al., 2011). A well-suited period to inves- tigate the underlying mechanisms by model simulations is the last deglaciation (Knorr and Lohmann, 2007; Liu et al., 2009; Menviel et al., 2011) due to the abundance of avail- able data based reconstructions (e.g. Lea et al., 2003; Mc- Manus et al., 2004; Peltier, 2004; Ahn and Brook, 2008; Gherardi et al., 2009). One of the most fundamental is- sues in this respect is the deﬁnition of a climate state to be used as an initial state. On account of the abundance of The simulated ocean state with the most realistic AMOC is characterized by a pronounced vertical stratiﬁcation, in line with reconstructions. Hosing experiments further sug- gest that the response of the glacial ocean is dependent on the ocean background state, i.e. only the state with robust strat- iﬁcation shows an overshoot behavior in the North Atlantic. We propose that the salinity stratiﬁcation represents a key control on the AMOC pattern and its transient response to perturbations. Furthermore, additional experiments suggest that the stratiﬁed deep ocean formed prior to the LGM dur- ing a time of minimum obliquity (∼27 ka BP). This indicates study. A summary of the experiment characteristics is also provided in Table 1. study. A summary of the experiment characteristics is also provided in Table 1. Published by Copernicus Publications on behalf of the European Geosciences Union. 1) (Braconnot et al., 2007; Weber et al., 2007). Thus, it is open to question whether the different LGM AMOC states are potentially as- sociated with different regimes due to the mean deep-ocean properties. Following this, we further elaborate the transient responses upon different background conditions. 2 Model and experimental design 26 Figure 1 Meridional section of zonal mean salinity in Atlantic Ocean for six PMIP2 models (CCSM3 (Otto-Bliesner et al., 2006) HadCM3M2 (Gordon et al., 2000), MIROC 3.2 (K-1-Model-Developers, 2004), ECBilt-CLIO (de Fig. 1. Meridional section of zonal mean salinity in Atlantic Ocean for six PMIP2 models (CCSM3 (Otto-Bliesner et al., 2007) HadCM3M2 (Gordon et al., 2000), MIROC 3.2 (K-1-Model- Developers, 2004), ECBilt-CLIO (de Vries and Weber, 2005), FGOALS-1.0g (Yu et al., 2002, 2004) and IPSL-CM4-V1-MR (Marti et al., 2005)). The stratiﬁcation in CCSM3 and HadCM3M2 is comparable with reconstruction (Otto-Bliesner et al., 2007), while the ocean structure in MIROC 3.2, ECBilt-CLIO, FGOALS- 1.0g and IPSL-CM4-V1-MR is more like the present-day, with the saltiest deep-water mass in the North Atlantic. According to their salinity structure, one can divide the PMIP2 models into two main classes, which are related to a highly stratiﬁed ocean, but weaker AMOC (i.e. CCSM3 and HadCM, as our quasi-equilibrium ocean state) and weaker stratiﬁed, but stronger AMOC (transient ocean state). 2.2.1 LGM simulations External forcing and boundary conditions are imposed ac- cording to the PMIP3 protocol for the LGM (available at http://pmip3.lsce.ipsl.fr/). The respective boundary condi- tions for the LGM comprise orbital forcing, greenhouse gas concentrations (CO2 = 185 ppm; N2O = 200 ppb; CH4 = 350 ppb), ocean bathymetry, land surface topography, run- off routes according to PMIP3 ice sheet reconstruction and Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. X. Zhang et al.: LGM simulations and implications for deglaciation et al.: LGM simulations and implications for deglaciation 26 Figure 1 Meridional section of zonal mean salinity in Atlantic Ocean for six PMIP2 models (CCSM3 (Otto-Bliesner et al., 2006) HadCM3M2 (Gordon et al., 2000), MIROC 3.2 (K-1-Model-Developers, 2004), ECBilt-CLIO (de Fig. 1. Meridional section of zonal mean salinity in Atlantic Ocean for six PMIP2 models (CCSM3 (Otto-Bliesner et al., 2007) HadCM3M2 (Gordon et al., 2000), MIROC 3.2 (K-1-Model- Developers, 2004), ECBilt-CLIO (de Vries and Weber, 2005), FGOALS-1.0g (Yu et al., 2002, 2004) and IPSL-CM4-V1-MR (Marti et al., 2005)). The stratiﬁcation in CCSM3 and HadCM3M2 is comparable with reconstruction (Otto-Bliesner et al., 2007), while the ocean structure in MIROC 3.2, ECBilt-CLIO, FGOALS- 1.0g and IPSL-CM4-V1-MR is more like the present-day, with the saltiest deep-water mass in the North Atlantic. According to their salinity structure, one can divide the PMIP2 models into two main classes, which are related to a highly stratiﬁed ocean, but weaker AMOC (i.e. CCSM3 and HadCM, as our quasi-equilibrium ocean state) and weaker stratiﬁed, but stronger AMOC (transient ocean state). 2320 et al.: LGM simulations and implications for deglaciation available proxy data and maximum ice sheet volume (Dup- lessy et al., 1988; Bard et al., 2000; Adkins et al., 2002; Pﬂau- mann, 2003; Peltier, 2004; Gersonde et al., 2005; Lynch- Stieglitz et al., 2007; Clark et al., 2009; Gutjahr and Lip- pold, 2011; Hesse et al., 2011), the Last Glacial Maximum (LGM, about 21 000 yr before present, hereafter 21 ka BP) commonly serves as the starting point for simulations of the last deglaciation (Liu et al., 2009; Menviel et al., 2011). Fur- thermore, it is also an excellent test bed for climate models to simulate a climate that strongly deviates from our mod- ern condition (e.g. the Paleoclimate Modeling Intercompar- ison Projection, or PMIP; Braconnot et al., 2007) for the future projection (Braconnot et al., 2012). However, there was a substantial difference in AMOC states among differ- ent models during the LGM (Otto-Bliesner et al., 2007). It is worth noting that PMIP utilized no speciﬁc protocol con- cerning the initial ocean condition for LGM simulations, and only CCSM3 and HadCM3M2, initialized from a previous glacial ocean state, were found to yield a simulated glacial ocean comparable to reconstructions (Fig. 2.1 Model description The comprehensive climate model COSMOS (ECHAM5- JSBACH-MPIOM) is used to analyse the different responses of the LGM to initial ocean states. The atmosphere model ECHAM5 (Roeckner et al., 2003), complemented by land surface model JSBACH (Brovkin et al., 2009), was used at T31 resolution (∼3.75◦) with 19 vertical layers. The ocean model MPI-OM (Marsland et al., 2003), including the dy- namics of sea ice formulated using viscous-plastic rheology (Hibler III, 1979), has the resolution of GR30 (∼3◦) in the horizontal and 40 uneven vertical layers. Note that the Hy- drological Discharge model (HDmodel) (Hagemann, 2002) is also embedded in the ECHAM5, guaranteeing a closure freshwater balance in our coupled system. The climate model was already utilized to analyse the last millennium (Jung- claus et al., 2010), warm climates in the Miocene (Knorr et al., 2011) and the Pliocene (Stepanek and Lohmann, 2012; Dowset et al., 2013), glacial (Kageyama et al., 2013; Gong et al., 2013) and interglacial climate (Varma et al., 2012; Wei et al., 2012; Wei and Lohmann, 2012). 2.2 Experimental design In the following section we describe the experimental set-up of the ten model simulations that represent the basis for our Clim. Past, 9, 2319–2333, 2013 www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ X. Zhang et al.: LGM simulations and implications for deglaciation Table 1. Experimental design of the simulations in this study. Integration Boundary conditions Initial conditions of model years PI (control) Pre-industrial Present-day ocean 3000 LGM2PI Pre-Industrial Glacial ocean 3000 LGMW 21 ka Glacial ocean 4000 LGMS 21 ka Present-day ocean 5000 LGMS27ka 21 ka, except 27 ka model year 4000 in LGMS 700 orbital forcing LGMW-0.2Sv 21 ka LGMW-e (model year 2900 in LGMW) 150 LGMW-1Sv 21 ka LGMW-e (model year 2900 in LGMW) 100 LGMS-0.2Sv 21 ka LGMS-tdeep (model year 2900 in LGMS) 150 LGMS-1Sv 21 ka LGMS-tdeep (model year 2900 in LGMS) 100 LGMS-e-0.2Sv 21 ka LGMS-e (model year 4600 in LGMS) 150 X. Zhang et al.: LGM simulations and implications for deglaciation 2321 increased global salinity (+1 psu compared to modern value) to account for a sea level drop of ∼116 m. years 2700 and 4500, respectively. In LGMW, due to the fact that there is almost no change in the climatology after model year 2700 (Figs. 2–7 and S2), the climatologically an- nual mean of model years 2900–3000 was chosen to repre- sent the quasi-equilibrium ocean state LGMW-e. In LGMS, there is almost no difference between climatology of model years 4600–4700 and 4500–5000. To better compare with the outputs from the 27 ka simulation (see details in Sect. 2.2.4), thus we deﬁne the climatology mean between model years 4600–4700 as LGMS-e to represent the quasi-equilibrium state in LGMS. Using the same LGM boundary conditions we performed two experiments, LGMW and LGMS, with different initial ocean states and integrated them for 4000 and 5000 yr, re- spectively. LGMS is initialized from a ocean state with im- posed present-day temperature and salinity ﬁelds (Levitus et al., 1998), while LGMW is initialized from a glacial ocean state. The initial glacial ocean was generated through an ocean-only model, MPI-OM (ocean component of our COS- MOS set-up), which was run for 3000 yr under the LGM con- ditions. 2.2 Experimental design To generate the glacial ocean state with MPI-OM, we obtained its atmospheric forcing from an ECHAM5 ex- periment in T31 resolution using SST forcing as provided by CLIMAP (CLIMAP, 1981) in a similar way as done with the older ECHAM3 version (Lohmann and Lorenz, 2000), and derived its initial ocean state and surface salinity restoring from PMIP2 model outputs of CCSM3 (the National Center for Atmospheric Research CCSM3 model) that is assumed to have a good performance on simulating the LGM climate state in comparison to other PMIP2 models (Otto-Bliesner et al., 2007; Weber et al., 2007). Note that the ocean state between model years 2500–3000 in LGMS also meets PMIP criteria (Fig. S1, for a zoom- in plot of AMOC indices and global mean SST), although the trend in its deep ocean properties is signiﬁcant (Fig. 4). Accordingly, to better compare with LGMW-e, model years 2900–3000 in LGMS are averaged to represent this ocean state and named as LGMS-tdeep here. p A comparison among the ocean states LGMS-tdeep, LGMS-e and LGMW-e was made with respect to the cor- responding climatology (Figs. 5–7 and S2) and surface and deep ocean trends (Figs. 2–4). It suggests that due to their similarity LGMS-e and LGMW-e can represent the ﬁnal equilibrium LGM state in our model, however LGMS-tdeep is the state in the transient phase of bottom-water properties. In this study, we will mainly focus on the contrast between LGMS-tdeep and LGMW-e (Figs. 2–4). To deﬁne the representative climatology from both LGM runs we employed the quasi-equilibrium criteria of the PMIP protocol (Braconnot et al., 2007) to assess the stability of the simulated ocean states. That is, quasi-equilibrium state can be deﬁned, as the global SST trend is less than 0.05 k century−1 as well as a stable AMOC. Figures 2 and 3 show the AMOC indices and 100 yr running means of global mean sea surface temperature (SST) for the simula- tions LGMW and LGMS. Compared to the gradual increase of AMOC and SST in LGMW, the decreasing trend in LGMS is particularly pronounced, especially after the model year 3000. 2.2.2 Pre-industrial simulations To examine whether the feature of time-dependency on ini- tial ocean states in glacial simulations is also present in a warm climate, we conducted two pre-industrial (PI) simula- tions in this study. One was initialized from the same present- day ocean state as LGMS, which is referred to as PI control run and has been analyzed by Wei et al. (2012). The other one, LGM2PI, was initialised from the glacial ocean state of Based on the PMIP criteria (Braconnot et al., 2007), LGMW and LGMS are in quasi-equilibrium after model www.clim-past.net/9/2319/2013/ Clim. Past, 9, 2319–2333, 2013 g et al.: LGM simulations and implications for deglaciation 2500 3000 3500 4000 4500 14.8 14.85 14.9 14.95 15 15.05 15.1 15.15 15.2 15.25 15.3 15.35 15.4 model year Global SST (degC) LGMW LGMS Figure 3 100-year running mean for global mean SST in LGMW (blue) LGMS (red). The gap between the model year 3000-3200 in LGMS is d missed dataset Units: °C Fig. 3. 100 yr running mean for global mean SST in LGMW (blue) and LGMS (red). The gap between the model year 3000–3200 in LGMS is due to missed dataset. Units: ◦C. ng et al.: LGM simulations and implications for deglaciation 2500 3000 3500 4000 4500 14.8 14.85 14.9 14.95 15 15.05 15.1 15.15 15.2 15.25 15.3 15.35 15.4 model year Global SST (degC) LGMW LGMS Figure 3 100-year running mean for global mean SST in LGMW (blue LGMS (red). The gap between the model year 3000-3200 in LGMS is d missed dataset Units: °C Fig. 3. 100 yr running mean for global mean SST in LGMW (blue) and LGMS (red). The gap between the model year 3000–3200 in LGMS is due to missed dataset. Units: ◦C. X. Zhang et al.: LGM simulations and implications for deglaciation 2322 2000 2500 2700 3000 3500 4000 4500 5000 15 20 25 30 35 model year AMOC index (Sv) LGM−W LGM−S a)# Fig.%2% 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 AABW−cell index (Sv) LGM−W 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 model year AABW−cell index (Sv) LGM−S b)% c)% Figure 2 AMOC indices with respect to a) NADW-c maximum value of stream function below 500m in the b-c) AABW-cell (defined as the maximum of absolu function below 2500m along 30 S) for LGMS (red) and Fig. 2. 2.2.4 The 27 ka simulation (defined as the As explicitly discussed in Sect. 3.2, reconstructions suggest that the recorded LGM ocean structure might have been formed prior to the LGM. To gain a deeper understanding on the inﬂuence of boundary conditions (especially insolation) in shaping the state of the LGM ocean and AMOC, we per- formed the simulation LGMS27ka in which orbital parame- ters of 27 ka BP (precession: 196.532◦, obliquity: 22.2514◦, eccentricity: 0.017451) (Laskar et al., 2004) are imposed on LGM boundary conditions. This simulation was initialized from model year 4000 of LGMS and is integrated for 700 yr (Table 1). The last 100 yr average was considered to represent the corresponding 27 ka BP climatology. .%2% 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 model year AABW−cell index (Sv LGM−S c)% Figure 2 AMOC indices with respect to a) NADW-ce maximum value of stream function below 500m in the N b-c) AABW-cell (defined as the maximum of absolut function below 2500m along 30 S) for LGMS (red) and L Fig. 2. AMOC indices with respect to (a) NADW-cell (deﬁned as the maximum value of stream function below 500 m in the North Atlantic) and (b–c) AABW-cell (deﬁned as the maximum of abso- lute value of stream function below 2500 m along 30◦S) for LGMS (red) and LGMW (blue). The bold solid lines are the 10 yr running mean. Units: Sv=106 m3 s−1. 2.2.2 Pre-industrial simulations AMOC indices with respect to (a) NADW-cell (deﬁned as the maximum value of stream function below 500 m in the North Atlantic) and (b–c) AABW-cell (deﬁned as the maximum of abso- lute value of stream function below 2500 m along 30◦S) for LGMS (red) and LGMW (blue). The bold solid lines are the 10 yr running mean. Units: Sv=106 m3 s−1. 2000 2500 2700 3000 3500 4000 4500 5000 15 20 25 30 35 model year AMOC index (Sv) LGM−W LGM−S a)# Figure 3 100-year running mean for global mean SST in LGMW (blue LGMS (red). The gap between the model year 3000-3200 in LGMS is d i d d t t U it °C Fig. 3. 100 yr running mean for global mean SST in LGMW (blue) and LGMS (red). The gap between the model year 3000–3200 in LGMS is due to missed dataset. Units: ◦C. 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 AABW−cell index (Sv) LGM−W b)% 3.1.1 Surface properties 28 LGMW. Both PI simulations were integrated for 3000 yr (Ta- ble 1) using identical PI boundary conditions as in previous studies in PMIP (Cruciﬁx et al., 2005). The average of the model years 2900–3000 is considered to represent the clima- tology in both simulations. 28 LGMW. Both PI simulations were integrated for 3000 yr (Ta- ble 1) using identical PI boundary conditions as in previous studies in PMIP (Cruciﬁx et al., 2005). The average of the model years 2900–3000 is considered to represent the clima- tology in both simulations. The global climatological mean SST are 14.9 ◦C and 15.3 ◦C in LGMW-e and LGMS-tdeep, i.e. 2.8 ◦C and 2.4 ◦C lower than the PI control run, respectively. The SST differences rel- ative to PI are similar in the quasi-equilibrium ocean states (Figs. 5 and 6). In the high latitudes of the Southern Hemi- sphere, our model simulates a pronounced annual mean cool- ing of SST around Antarctica (Figs. 5 and 6), in line with proxy data (Gersonde et al., 2005). In the Northern Hemi- sphere, a robust meridional thermal gradient is well simu- lated around 40 ∼45◦N, and the most pronounced cooling is found off the eastern coast of Iceland to eastern part of Nordic Sea (Figs. 5 and 6). Both features are comparable to reconstructions (Kucera et al., 2005; de Vernal et al., 2006). In contrast to the MARGO data (Waelbroeck et al., 2009), X. Zhang et al.: LGM simulations and implications for deglaciation 2323 b)# d)# a)# c)# LGMS-t LGMS-e LGMW-e LGMW (3800-4000) Figure 4 Salinity trend in Atlantic Ocean for model year 2800-3000 in LGMS-td (A) and LGMW-e (C) and model year 4500-4700 in LGMS-e Fig. 4. Salinity trend in Atlantic Ocean for model year 2800–3000 in LGMS-tdeep (A) and LGMW-e (C) and model year 4500–4700 in LGMS-e (B) and model year 3800–4000 in LGMW (D). Units: psu century−1. Figure 4 Salinity trend in Atlantic Ocean for model year 2800-3000 in LGMS-tdeep (A) and LGMW-e (C) and model year 4500-4700 in LGMS-e Fig. 4. Salinity trend in Atlantic Ocean for model year 2800–3000 in LGMS-tdeep (A) and LGMW-e (C) and model year 4500–4700 in LGMS-e (B) and model year 3800–4000 in LGMW (D). Units: psu century−1. W (D). Units: psu/century. are consistent with the reconstructions, representing the cli- matological surface patterns during the LGM. W (D). Units: psu/century. are consistent with the reconstructions, representing the cli- matological surface patterns during the LGM. (B) and model year 3800-4000 in LG our model as well as PMIP2 models underestimate the pro- nounced east–west SST anomaly gradient in the northern North Atlantic. Despite the different initial conditions in LGMW and LGMS, there is also a reasonable agreement between the sea ice concentrations (SIC) in both ocean states and proxy data (Fig. 5a, b), such as the austral winter sea ice extent in the At- lantic sector and the austral summer sea ice extent in the In- dian ocean sector (Gersonde et al., 2005). But the simulations underestimate the large extent of summer sea ice between 5◦E and 5◦W in the Southern Ocean. During boreal winter, sea ice increases, especially along the coast of Newfound- land, extending far into the western Atlantic (Pﬂaumann, 2003; Kucera et al., 2005; de Vernal et al., 2006). Sea ice ex- tent is underestimated in the north-eastern Atlantic Ocean in our model (Paul and Schäfer-Neth, 2003; Pﬂaumann, 2003) due to an active North Atlantic current that maintains rel- atively warm conditions at the sea surface (Fig. 5). During boreal summer the eastern part of the Nordic seas is partly sea ice-free (Fig. 5a, b), which is spatially coherent with sea ice free conditions as indicated in the GLAMAP reconstruc- tion of the LGM (Paul and Schäfer-Neth, 2003; Pﬂaumann, 2003). X. Zhang et al.: LGM simulations and implications for deglaciation In addition, there is perennial summer sea ice extent in the west of the Nordic Sea along the eastern coast of Green- land and Labrador Sea (Fig. 5), in agreement with the recon- structions (Pﬂaumann, 2003; Kucera et al., 2005; de Vernal et al., 2006). In summary, the similar surface properties gen- erated in both LGM ocean states LGMS-tdeep and LGMW-e 2.2.3 Hosing experiments To investigate the stability of the two LGM ocean states and the dependence of the AMOC on ocean stratiﬁcation, we have imposed constant freshwater perturbations (FWP) of +0.2 Sv and +1 Sv over the Ice-Rafted Debris belt (around 40◦N–55◦N, 45◦W–20◦W) (Hemming, 2004) in the North Atlantic for 150 and 100 yr, respectively (Table 1). After the FWP, the experiments continued running for another 250 yr to evaluate the recovery processes. www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ Clim. Past, 9, 2319–2333, 2013 X. Zhang et al.: LGM simulations and implications for deglaciation 3.1.2 Distinct deep ocean properties Figure 7 shows the meridional sections of zonal mean sea salinity and temperature along the Atlantic Ocean and the spatial patterns of the AMOC. In terms of the water mass properties of ocean interior there are pronounced differences between LGMW-e and LGMS-tdeep (Fig. 7). Only LGMW- e possesses an important key feature of the glacial ocean, i.e. the saltier and colder AABW at the bottom of the South- ern Ocean compared to LGMS-tdeep. This is consistent with a reconstruction of Adkins et al. (2002), while the wa- ter mass of LGMS-tdeep is more similar to the present-day ocean state (Fig. 7e, f). According to water mass conﬁgu- ration reconstructed from nutrient tracers (Duplessy et al., 1988; Curry and Oppo, 2005; Marchitto and Broecker, 2006; Lynch-Stieglitz et al., 2007; Hesse et al., 2011), the North At- lantic Deep Water (NADW) shoals to about 2000–2500 m as Glacial North Atlantic Intermediate Water (GNAIW) due to the enhanced northward invasion of Antarctic Bottom Water (AABW) at the LGM. The AMOC associated with the sink- ing of NADW (hereafter NADW-cell) in LGMW-e shoals by ∼500 m relative to present-day to 2500 m. This is indicative of a shallow NADW-cell and an abyssal ocean occupied by the AABW (hereafter AABW-cell) (Fig. 7a, d, g). A similar pattern is also found in the quasi-equilibrium state LGMS-e www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ Clim. Past, 9, 2319–2333, 2013 X. Zhang et al.: LGM simulations and implications for deglaciation 2324 p g Figure 6 Anomaly of annual mean SST for a) LGM LGMW-e minus LGMS-tdeep and c) LGMW-e minus LG a) b) c) Fig. 6. Anomaly of annual mean SST for (a) LGMW-e minus PI, (b) LGMW-e minus LGMS-tdeep and (c) LGMW-e minus LGMS- e. Units: ◦C. Figure 5 Annual mean of sea surface temperature (SST, unit: °C, shaded) and seasonal sea ice concentration (SIC, unit: %, contour) in the LGMW-e (a) and LGMS-tdeep (b). The white lines represent winter for each hemisphere, while the red line represents summer. The dashed lines indicate 15% SIC, and solid lines 90% SIC. (c) Zonal mean of global SST in PI (black), LGMW-e (blue) and LGMS-t (red). Units: °C. a) b) c) Fig. 5. Annual mean of sea surface temperature (SST, unit: ◦C, shaded) and seasonal sea ice concentration (SIC, unit: %, contour) in the LGMW-e (a) and LGMS-tdeep (b). 3.1.2 Distinct deep ocean properties The white lines repre- sent winter for each hemisphere, while the red line represents sum- mer. The dashed lines indicate 15 % SIC, and solid lines 90% SIC. (c) Zonal mean of global SST in PI (black), LGMW-e (blue) and LGMS-tdeep (red). Units: ◦C. a) c) Figure 5 Annual mean of sea surface temperature (SST b) b) g p ( ) and seasonal sea ice concentration (SIC, unit: %, contour) in the LGMW-e (a) and LGMS-tdeep (b). The white lines represent winter for each hemisphere, while the red line represents summer. The dashed lines indicate 15% SIC, and solid lines 90% SIC. (c) Zonal mean of global SST in PI (black), LGMW-e (blue) and LGMS-t (red). Units: °C. Fig. 5. Annual mean of sea surface temperature (SST, unit: ◦C, shaded) and seasonal sea ice concentration (SIC, unit: %, contour) in the LGMW-e (a) and LGMS-tdeep (b). The white lines repre- sent winter for each hemisphere, while the red line represents sum- mer. The dashed lines indicate 15 % SIC, and solid lines 90% SIC. (c) Zonal mean of global SST in PI (black), LGMW-e (blue) and LGMS-tdeep (red). Units: ◦C. c) 31 (Fig. S2). However, in LGMS-tdeep the NADW-cell pene- trates to ∼3100 m, even deeper than today (Fig. 7b, e, h). trates to ∼3100 m, even deeper than today (Fig. 7b, e, h). Besides the evident contrast in the deep ocean properties, differences in the AMOC strength between LGMW-e and LGMS-tdeep are also pronounced, although both are stronger than present-day (Fig. 6g, h, i). In our LGM simulations, enhanced southern westerlies relative to the PI control run (Fig. S3) result in a stronger NADW-cell due to a stronger “Drake Passage Effect” via the enhanced Ekman upwelling of the deep water (Toggweiler and Samuels, 1995; Wei et al., 2012). Furthermore, stronger net evaporation in the Atlantic catchment area (Fig. S4) combined with more heat loss to the atmosphere from the convection sites over the North At- lantic (Fig. S5) also result in an enhanced NADW-cell (We- ber et al., 2007). In addition, the formation of AABW as a result of brine rejection during sea ice formation is enhanced due to extensive sea ice formation and increased sea ice ex- port during the LGM (Fig. S6) (Shin et al., 2003). As a con- sequence, the expected stronger AMOC states in LGMW- e and LGMS-tdeep should be distinct from today. X. Zhang et al.: LGM simulations and implications for deglaciation 2325 a) b) c) d) e) f) g) h) i) LGMW-e LGMS-t PI control LGMW-e LGMS-t PI control LGMW-e LGMS-t PI control Figure 7. Meridional section of zonal mean temperature (A-C, units: °C), salinity (D-F, units: psu) and stream function (G-I, units: Sv (106 m3/s)) in Fig. 7. Meridional section of zonal mean temperature ((A–C), units: ◦C), salinity ((D–F), units: psu) and stream function ((G–I), units: Sv (106 m3 s−1)) in the Atlantic Ocean. For panel (F), we added 1 psu to the salinity ﬁeld for a better comparison with the glacial salinity structure. a) b LGMW-e d) e LGMW-e LGMS-t f) d) e) h) i) LGMS-t PI control g) h LGMW-e g) Figure 7. Meridional section of zonal mean temperature (A-C, units: °C), salinity (D-F, units: psu) and stream function (G-I, units: Sv (106 m3/s)) in Fig. 7. Meridional section of zonal mean temperature ((A–C), units: ◦C), salinity ((D–F), units: psu) and stream function ((G–I), units: Sv (106 m3 s−1)) in the Atlantic Ocean. For panel (F), we added 1 psu to the salinity ﬁeld for a better comparison with the glacial salinity structure. the Atlantic Ocean. For panel (F), w better comparison with the glacial sal The different ocean states found in our model are in quasi- equilibrium according to the PMIP criteria but possess dis- tinct features with respect to ocean structure and overturn- ing circulation, which can be also found in PMIP2 models (Fig. 1). Accordingly, one can classify the simulated ocean states in PMIP2 models into two classes, “glacial-like” ocean state as LGMW-e and “present-day-like” glacial ocean state as LGMS-tdeep. added 1 psu to the salinity field for a ty structure. ocean state to the other (Fig. 8). This implies that the rea- son for the different glacial ocean states is not related to the hydrological balance of the North Atlantic, but to the mean deep-ocean properties that exist due to different ini- tial ocean conditions. After the FWP, the overshoot of the AMOC in LGMW-e (see LGMS-e case in Fig. S7) results in an abrupt warming over Greenland for several decades, but not in the LGMS-tdeep case (Figs. 8 and 9). Given the dis- tinct ocean structures during and after the FWP, we propose that the stratiﬁed glacial ocean plays an important role in the AMOC recovery by inﬂuencing the subsurface warming in convection sites (especially in the northern North Atlantic, Fig. 3.1.3 Distinct AMOC recovery processes to freshwater perturbation Previous model studies show a bistable regime of the AMOC in its parameter space under present climate conditions, whereby North Atlantic deep-water formation can be “on” (as in the present climate) or “off” (e.g., Rahmstorf et al., 2005). As a consequence different AMOC states can exist within the same boundary conditions, depending on the ini- tial conditions in the ocean. In addition, transitions between different ocean states can be fulﬁlled by modifying the hy- drological balance in the Atlantic basin, i.e. the so-called hosing experiment (e.g. Rahmstorf et al., 2005). To inves- tigate the potential transition between ocean states LGMW- e and LGMS-tdeep, Fig. 8 shows the time series of AMOC indices for respective hosing experiments. It is notable that neither of these are able to trigger the transition from one X. Zhang et al.: LGM simulations and implications for deglaciation S8), northward transport of tropical warmer and saltier water and basin-wide salinity adjustment (Figs. S9 and S10) (cf. Mignot et al., 2007; Liu et al., 2009; Cheng et al., 2011). This may explain the abrupt warming events over Greenland following the Heinrich events during glacial periods (e.g. the abrupt warming after Heinrich Event 2) (Dansgaard et al., 1993; Blunier and Brook, 2001). 3.1.2 Distinct deep ocean properties Note that the overturning circulation is evidently reduced due to the stronger vertical stratiﬁcation that weakens the AMOC from ∼27 Sv in LGMS-tdeep (Fig. 7h) to ∼18 Sv in LGMW-e (Fig. 7g). Furthermore, the resulting AMOC in LGMW-e is also supported by reconstructions, suggesting that the glacial Figure 6 Anomaly of annual mean SST for a) LGM LGMW-e minus LGMS-tdeep and c) LGMW-e minus LG Fig. 6. Anomaly of annual mean SST for (a) LGMW-e minus PI, (b) LGMW-e minus LGMS-tdeep and (c) LGMW-e minus LGMS- e. Units: ◦C. 32 AMOC is shallower (Duplessy et al., 1988; Curry and Oppo, 2005; Marchitto and Broecker, 2006) but as strong as in the subsequent warm periods within data uncertainties (Lippold et al., 2012; Ritz et al., 2013). Although proxy data for the LGM are actually consistent with a range of Atlantic circula- tion states (McCave et al., 1995; Yu et al., 1996; McManus et al., 2004; Lynch-Stieglitz et al., 2007; Praetorius et al., 2008; Gherardi et al., 2009; Huybers and Wunsch, 2010), even in- cluding the modern state (e.g., LeGrand and Wunsch, 1995), the ocean state LGMS-tdeep can be ruled out due to its large inconsistency with the proxy data. www.clim-past.net/9/2319/2013/ Clim. Past, 9, 2319–2333, 2013 X. Zhang et al.: LGM simulations and implications for deglaciation www.clim-past.net/9/2319/2013/ 3.1.4 Reconciling the discrepancies in simulated LGM ocean states Time series of AMOC in the 0.2 Sv (A: FWP lasts for 150 yr) and the 1Sv (B: FWP lasts for 100 yr) hosing experiments LGMW-e (blue) and LGMS-tdeep (red), respectively. The hosing experiments started from the model year 2700 in each LGM simulation. Dashed lines represent the LGM control runs (A), and solid lines the hos- ing experiments (A, B). Despite the different amount of FWP, a transition between the states LGMW-e and LGMS-tdeep is not trig- gered. A robust overshoot of the AMOC is only detected in quasi- equilibrium ocean states (see also Fig. S7). 0 100 200 300 400 −35 −30 −25 −20 −15 −10 year SAT Greenland surface temperature LGMW−0.2Sv LGMS−0.2Sv A B Figure 9 A) Surface air temperature (SAT) in the latit the North Atlantic in LGMW-0.2Sv (blue) and LGM running mean was used to filter out the high frequenc The red and blue dashed lines indicate the pre-hosi Fig. 9. (A) Surface air temperature (SAT) in the latitude belt of 60– 70◦N in the North Atlantic in LGMW-0.2Sv (blue) and LGMS- 0.2Sv (red). 5 yr running mean was used to ﬁlter out the high- frequency signals of the SAT. The red and blue dashed lines indicate the pre-hosing reference level for LGMS-tdeep and LGMW-e, re- spectively. (B) Excess surface temperature increase related to the overshoot of the AMOC (270th–300th yr) in LGMW-0.2Sv. The d t t i l ti t LGMW i th N th 0 100 200 300 400 −35 −30 −25 −20 −15 −10 year SAT Greenland surface temperature LGMW−0.2Sv LGMS−0.2Sv A −300 −150 0 150 300 450 600 0 5 10 15 20 25 30 35 year AMOC index (Sv) Freshwater Perturbation LGM−W LGM−S LGMW−0.2Sv LGMS−0.2Sv A Greenland surface temperature Freshwater Perturbation A lasts for 150 years) periments LGMW e A B 0 100 200 300 400 0 5 10 15 20 25 30 35 Year Sv Freshwater Perturbation LGMW-1Sv LGMS-1Sv B Freshwater Perturbation Figure 8 Time series of AMOC in the 0.2Sv (A: FW and the 1Sv (B: FWP lasts for 100 years) hosing (blue) and LGMS-tdeep (red), respectively. The hos from the model year 2700 in each LGM simulation the LGM control runs (A), and solid lines the hosi Despite the different amount of FWP, a transiti Fig. 8. 3.1.4 Reconciling the discrepancies in simulated LGM ocean states Time series of AMOC in the 0.2 Sv (A: FWP lasts for 150 yr) and the 1Sv (B: FWP lasts for 100 yr) hosing experiments LGMW-e (blue) and LGMS-tdeep (red), respectively. The hosing experiments started from the model year 2700 in each LGM simulation. Dashed lines represent the LGM control runs (A), and solid lines the hos- ing experiments (A, B). Despite the different amount of FWP, a transition between the states LGMW-e and LGMS-tdeep is not trig- gered. A robust overshoot of the AMOC is only detected in quasi- equilibrium ocean states (see also Fig. S7). B B g experiments started ashed lines represent experiments (A, B). between the states ershoot of the AMOC lso Fig. S7). Figure 9 A) Surface air temperature (SAT) in the the North Atlantic in LGMW-0.2Sv (blue) and running mean was used to filter out the high freq The red and blue dashed lines indicate the pre- LGMS-tdeep and LGMW-e, respectively. B) Ex increase related to the overshoot of the AMO Fig. 9. (A) Surface air temperature (SAT) in the latitude belt of 60– 70◦N in the North Atlantic in LGMW-0.2Sv (blue) and LGMS- 0.2Sv (red). 5 yr running mean was used to ﬁlter out the high- frequency signals of the SAT. The red and blue dashed lines indicate the pre-hosing reference level for LGMS-tdeep and LGMW-e, re- spectively. (B) Excess surface temperature increase related to the overshoot of the AMOC (270th–300th yr) in LGMW-0.2Sv. The pronounced temperature increase relative to LGMW in the North Hemisphere is up to 6.8 ◦C at convection sites in the Northern At- lantic. 34 is only detected in quasi-equilibrium ocean states (se (e.g. LGMS), the simulated ocean state after 2500 model years reaches a temporary quasi-equilibrium state identiﬁed by the PMIP protocol, in which the trends in the deep ocean are signiﬁcant. (Figs. 2–4 and S1). The simulated surface properties are consistent with reconstructions (Figs. 5 and 6), further masking the transient deep ocean characteristics. This feature is not identiﬁed in the simulation initialized from a glacial ocean state (LGMW). Due to the lack of a spec- iﬁcation regarding the initial ocean state for simulating the LGM, all the PMIP2 models (except CCSM3 and HadCM) were initialized from the present-day ocean (Braconnot et al., 2007; Weber et al., 2007). As a consequence, CCSM3 and HadCM eventually generate a glacial-like ocean (e.g. 3.1.4 Reconciling the discrepancies in simulated LGM ocean states A signiﬁcant feature in our LGM simulations is the different equilibrium timescales depending on the initial ocean states. When the present-day ocean serves as the initialization www.clim-past.net/9/2319/2013/ Clim. Past, 9, 2319–2333, 2013 et al.: LGM simulations and implications for deglaciation P lasts for 150 years) xperiments LGMW-e g experiments started ashed lines represent experiments (A, B). between the states ershoot of the AMOC lso Fig. S7). 0 100 200 300 400 −35 −30 −25 −20 −15 −10 year SAT Greenland surface temperature LGMW−0.2Sv LGMS−0.2Sv A B Figure 9 A) Surface air temperature (SAT) in the l the North Atlantic in LGMW-0.2Sv (blue) and L running mean was used to filter out the high frequ The red and blue dashed lines indicate the pre-h LGMS-tdeep and LGMW-e, respectively. B) Ex increase related to the overshoot of the AMO Fig. 9. (A) Surface air temperature (SAT) in the latitude belt of 60– 70◦N in the North Atlantic in LGMW-0.2Sv (blue) and LGMS- 0.2Sv (red). 5 yr running mean was used to ﬁlter out the high- frequency signals of the SAT. The red and blue dashed lines indicate the pre-hosing reference level for LGMS-tdeep and LGMW-e, re- spectively. (B) Excess surface temperature increase related to the overshoot of the AMOC (270th–300th yr) in LGMW-0.2Sv. The pronounced temperature increase relative to LGMW in the North Hemisphere is up to 6.8 ◦C at convection sites in the Northern At- lantic. X. Zhang et al.: LGM simulations and implications for deglaciation 2326 0 100 200 300 400 0 5 10 15 20 25 30 35 Year Sv Freshwater Perturbation LGMW-1Sv LGMS-1Sv B Figure 8 Time series of AMOC in the 0.2Sv (A: FWP lasts for 150 years) and the 1Sv (B: FWP lasts for 100 years) hosing experiments LGMW-e (blue) and LGMS-tdeep (red), respectively. The hosing experiments started from the model year 2700 in each LGM simulation. Dashed lines represent the LGM control runs (A), and solid lines the hosing experiments (A, B). Despite the different amount of FWP, a transition between the states LGMW e and LGMS td is not triggered A robust overshoot of the AMOC −300 −150 0 150 300 450 600 0 5 10 15 20 25 30 35 year AMOC index (Sv) Freshwater Perturbation LGM−W LGM−S LGMW−0.2Sv LGMS−0.2Sv A Fig. 8. 3.1.4 Reconciling the discrepancies in simulated LGM ocean states LGMW-e) as well as the AMOC overshoot after the FWP (e.g. in CCSM3, Cheng et al., 2011), whereas other simu- lations generate a present-day-like ocean (e.g. LGMS-tdeep) 35 LGMW-0.2Sv. The pronounced temperature incr the North Hemisphere is up to 6.8°C at conv Atlantic. without the feature of an AMOC overshoot (e.g. in ECBilt- CLIO-VECODE, Roche et al., 2010), emphasizing the im- portant role played by initial ocean states on LGM simula- tions (Figs. 1, 8 and 9). Furthermore, our results could be in- terpreted in the sense that the large spread of simulated LGM ocean state among the PMIP2 models can be attributed to different (or insufﬁcient) deep ocean equilibration or initial- ization. 3.1.5 Deep ocean quasi-equilibrium criteria It is noteworthy that the fundamental difference between LGM ocean states LGMW-e and LGMS-tdeep is their distinct www.clim-past.net/9/2319/2013/ Meridional section of zonal mean salinity anomalies (shaded) in global ocean between LGMS27ka and LGMS-e. For the comparison, we averaged the corresponding model year 4600–4700 in both experiments. 36 OS, black dashed). d) in Figure 12 Meridional section of zonal mea in the Atlantic Ocean in LGM2PI. The de t l i di ti th t th P i d t Fig. 12. Meridional section of zonal mean temperature (A) and salinity (B) in the Atlantic Ocean in LGM2PI. The deep ocean struc- ture is similar to PI control run, indicating that the Pre-industrial simulation is insensitive to the initial ocean condition. Figure 11 Meridional section of zonal mean salinity anomalies (sha global ocean between LGMS27ka and LGMS-e. For the comparis averaged the corresponding model year 4600-4700 in both experimen Fig. 11. Meridional section of zonal mean salinity anomalies (shaded) in global ocean between LGMS27ka and LGMS-e. For the comparison, we averaged the corresponding model year 4600–4700 in both experiments. initial ocean condition. equilibrium, an equilibrium criteria for deep ocean properties should be well speciﬁed for future model inter-comparisons. Shown in Fig. 4 are the salinity trends in the Atlantic Ocean in LGMS-tdeep, LGMS-e, LGMW-e and model years 3800–4000 of LGMW. In the quasi-equilibrium ocean states in LGMW and LGMS-e (Fig. 4b–d), salinity varies at a rate of no more than 0.006 psu century−1 at a water depth lower than 3000 m, whereas up to or even more than 0.01 psu century−1 in LGMS-tdeep (Fig. 4a). In addition, the deep ocean salinity trend is relatively larger in the Atlantic section of the Southern Ocean that is one of main formation sites of AABW. Therefore, we propose that the glacial deep initial ocean condition. equilibrium, an equilibrium criteria for deep ocean properties should be well speciﬁed for future model inter-comparisons. Shown in Fig. 4 are the salinity trends in the Atlantic Ocean in LGMS-tdeep, LGMS-e, LGMW-e and model years 3800–4000 of LGMW. In the quasi-equilibrium ocean states in LGMW and LGMS-e (Fig. 4b–d), salinity varies at a rate of no more than 0.006 psu century−1 at a water depth lower than 3000 m, whereas up to or even more than 0.01 psu century−1 in LGMS-tdeep (Fig. 4a). In addition, the deep ocean salinity trend is relatively larger in the Atlantic section of the Southern Ocean that is one of main formation sites of AABW. www.clim-past.net/9/2319/2013/ For the comparison, we averaged the corresponding model year 4600–4700 in both experiments. A B Figure 12 Meridional section of zonal mean t in the Atlantic Ocean in LGM2PI. The deep control run, indicating that the Pre-industrial initial ocean condition. Fig. 12. Meridional section of zonal mean temperature (A) and salinity (B) in the Atlantic Ocean in LGM2PI. The deep ocean struc- ture is similar to PI control run, indicating that the Pre-industrial simulation is insensitive to the initial ocean condition. equilibrium, an equilibrium criteria for deep ocean properties should be well speciﬁed for future model inter-comparisons Zonal mean SST anomaly between PMIP2 models and WOA98 −70 −65 −60 −55 −50 −45 −40 −4 −3 −2 −1 0 1 2 3 4 Latitude SST Zonal mean SST anomaly between PMIP2 models and WOA98 CCSM 3 MIROC 3.2 HadCM3M2 IPSL CM4 V1 ECBilt−CLIO FGOALs 1.0g COSMOS ∆"SST"()" A 10 Zonal mean of SST anomaly between respective PI control runs odels participating in PMIP2 and observational data (World Oce 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid Fig. 10. Zonal mean of SST anomaly between respective PI control runs of the models participating in PMIP2 and observational data (World Ocean Atlas 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M2 (green solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid), FGOALS-1.0g (cyan solid)), as well as the model used in this study (COSMOS, black dashed). 10 Zonal mean of SST anomaly between respective PI control runs odels participating in PMIP2 and observational data (World Ocea 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid Fig. 10. Zonal mean of SST anomaly between respective PI control runs of the models participating in PMIP2 and observational data (World Ocean Atlas 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M2 (green solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid), FGOALS-1.0g (cyan solid)), as well as the model used in this study (COSMOS, black dashed). 10 Zonal mean of SST anomaly between respective PI control runs Fig. 10. Zonal mean of SST anomaly between respective PI control B 36 MOS, black dashed). Figure 11 Meridional section of zonal mean salinity anomalies (sha global ocean between LGMS27ka and LGMS-e. For the comparis averaged the corresponding model year 4600-4700 in both experimen Fig. 11. www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ Clim. Past, 9, 2319–2333, 2013 X. Zhang et al.: LGM simulations and implications for deg −70 −65 −60 −55 −50 −45 −40 −4 −3 −2 −1 0 1 2 3 4 Latitude SST Zonal mean SST anomaly between PMIP2 models and WOA98 CCSM 3 MIROC 3.2 HadCM3M2 IPSL CM4 V1 ECBilt−CLIO FGOALs 1.0g COSMOS ∆"SST"()" e 10 Zonal mean of SST anomaly between respective PI control runs models participating in PMIP2 and observational data (World Ocea 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M n solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid ALS 1 0 ( lid)) ll th d l d i thi t d Fig. 10. Zonal mean of SST anomaly between respective PI control runs of the models participating in PMIP2 and observational data (World Ocean Atlas 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M2 (green solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid), FGOALS-1.0g (cyan solid)), as well as the model used in this study (COSMOS, black dashed). X. Zhang et al.: LGM simulations and implications for deglaciation 2327 X. Zhang et al.: LGM simulations and implications for deglaciation 2327 36 −70 −65 −60 −55 −50 −45 −40 −4 −3 −2 −1 0 1 2 3 4 Latitude SST Zonal mean SST anomaly between PMIP2 models and WOA98 CCSM 3 MIROC 3.2 HadCM3M2 IPSL CM4 V1 ECBilt−CLIO FGOALs 1.0g COSMOS ∆"SST"()" re 10 Zonal mean of SST anomaly between respective PI control runs of models participating in PMIP2 and observational data (World Ocean s 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M2 en solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid), OALS-1.0g (cyan solid)), as well as the model used in this study SMOS, black dashed). Fig. 10. Zonal mean of SST anomaly between respective PI control runs of the models participating in PMIP2 and observational data (World Ocean Atlas 98), i.e. CCSM3 (black solid), MIROC 3.2 (red solid), HadCM3M2 (green solid), IPSL-CM4-V1-MR (blue solid), ECBilt-CLIO (yellow solid), FGOALS-1.0g (cyan solid)), as well as the model used in this study (COSMOS, black dashed). Figure 11 Meridional section of zonal mean salinity anomalies (shaded) in global ocean between LGMS27ka and LGMS-e. For the comparison, we averaged the corresponding model year 4600-4700 in both experiments. Fig. 11. Meridional section of zonal mean salinity anomalies (shaded) in global ocean between LGMS27ka and LGMS-e. www.clim-past.net/9/2319/2013/ Therefore, we propose that the glacial deep vertical stratiﬁcation associated with the AABW-cell, which is relatively stable compared with the NADW-cell in the LGM simulations (Fig. 2). This is supposed to be the main cause for a weaker NADW-cell associated with a pronounced vertical stratiﬁcation, owing to continuous transportation of the dense AABW to the abyssal Atlantic basin in LGMS. To explicitly diagnose the transient characteristics of the deep ocean in LGMS and qualify the possibility that the deep ocean in some of the PMIP2 models were not in quasi- www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ 38 Clim. Past, 9, 2319–2333, 2013 X. Zhang et al.: LGM simulations and implications for deglaciation 2328 A B C D Figure 13 Zonal mean temperature (A, C) and salinity (B, D) anomalies between the 350th and 200th model year in LGM2PI (A, B) and LGMS (C, Fig. 13. Zonal mean temperature (A, C) and salinity (B, D) anomalies between the 350th and 200th model year in LGM2PI (A, B) and LGMS (C, D). Note that the range of color bar in LGM2PI is twice as large as in LGMS. C A C D B B D Figure 13 Zonal mean temperature (A, C) and salinity (B, D) anomalies between the 350th and 200th model year in LGM2PI (A, B) and LGMS (C, Fig. 13. Zonal mean temperature (A, C) and salinity (B, D) anomalies between the 350th and 200th model year in LGM2PI (A, B) and LGMS (C, D). Note that the range of color bar in LGM2PI is twice as large as in LGMS. 40 . timescale for the PMIP2 models initialized from present-day ocean state could be no less than 5000 yr. Given the equilib- rium timescale of ∼2500 yr in LGMW, it is of utmost impor- tance to specify one standard glacial ocean state to initialize the glacial simulations in the new phase of PMIP for the im- provement of LGM simulations and future inter-model and model-data comparison. 39 LG ocean can be diagnosed as quasi-equilibrium at least when basin-wide average salinity at depths larger than 3000 m varies at a rate less than 0.006 psu century−1 in Atlantic Ocean and less than 0.008 psu century−1 in Atlantic section of Southern Ocean. Previous model studies suggested that the strengthened sea ice formation and export under a cold climate could enhance brine rejection in the Southern Ocean, leading to a strength- ened AABW (Shin et al., 2003; Butzin et al., 2005; Liu et al., 2005; Otto-Bliesner et al., 2007). This suggests that the colder the simulated Southern Ocean is, the more efﬁcient the AABW formation. Figure 10 shows the zonal mean SST bias of the PMIP2 models with observation data at present- day. www.clim-past.net/9/2319/2013/ It is evident that only CCSM3 in the PMIP2 models and the model used in this study (COSMOS) have a general cooling bias south of 50◦S that is close to the northern edge of winter sea ice cover during the LGM (Gersonde et al., 2005). This surface cooling bias may accelerate the forma- tion of AABW and thus shorten the equilibrium timescale for the deep ocean. Considering the integration time of 5000 yr in the simulation LGMS, we suggest that the equilibrium X. Zhang et al.: LGM simulations and implications for deglaciation of zonal mean sea salinity and temperature along Atlantic Ocean in simulation LGM2PI. The resulting deep ocean properties are similar to our PI control run (Fig. 12), implying the equilibrium timescale of the deep ocean under PI bound- ary conditions is shorter than under LGM conditions. northward invasion of AABW. However, it is worth noting that the nutrient proxy data cannot rule out that the recon- structed ocean structure during the LGM might have been formed prior to the LGM. Recently, Schmitt et al. (2012) suggested that the carbon cycle in the climate system during the LGM was already in an equilibrium and the net trans- fer of carbon to the deep ocean had occurred prior to the LGM. Additionally, in previous ocean model studies a glacial ocean comparable to the reconstructions can be generated only if sea ice export is imposed to the glacial Southern Ocean (Butzin et al., 2005; Hesse et al., 2011), suggesting that the deep-ocean water mass distribution as inferred from reconstructions is sensitive to the sea ice dynamics in the Southern Ocean. Using a sea ice reconstruction based on di- atoms, Allen et al. (2011) suggested that more extensive sea ice extent was found between ∼22 ka and ∼30 ka BP, over- lapping with the minimum temperature in Antarctica that is attributed to the lowest obliquity-reducing annual mean solar radiation to high latitudes. This indicates that the brine re- jection associated with sea ice formation might be stronger than during the LGM, resulting in a stronger AABW forma- tion. Notably, there is also a sharp decrease of atmospheric CO2 concentration and benthic δ13C at the beginning of Ma- rine Isotope Stage 2 (MIS2, ∼27 ka BP) (e.g. Hodell et al., 2003; Ahn and Brook, 2008), implicating abrupt formation of an abyssal carbon reservoir. In addition, the northward in- vasion of enhanced AABW, beginning at ∼27 ka BP, also in- dicates the formation of the reconstructed LGM ocean struc- ture (Gutjahr and Lippold, 2011) prior to the LGM. Accord- ingly, it is conceivable that the reconstructed water mass con- ﬁguration during the LGM might stem from the inception of MIS2 (∼27 ka BP) that might be the onset of the last deglaciation due to the following Heinrich Event 2 and sub- sequent abrupt warming over Greenland (Hemming, 2004; Blunier and Brook, 2001) and slight increase of atmospheric CO2 concentration (Ahn and Brook, 2008). 4 Summary and conclusions Based on our investigations of transient and quasi- equilibrium integrations of the glacial and interglacial cli- mate we ﬁnd a suite of key ﬁndings and conclusions that can be summarized in the context of (A) time-dependent charac- teristics and (B) general implications. (A) Time-dependent characteristics of ocean properties and the AMOC: – Climatological surface characteristics can be similar and quasi-stationary, even when a signiﬁcant trend in deep ocean properties still exists. – According to the PMIP criteria (Braconnot et al., 2007), such quasi-stationary states can be classi- ﬁed as equilibrium states, based on surface temper- ature trend analysis. Hence, caution on deep ocean must be taken when these allegedly quasi-equilibrium states, on the basis of surface properties, are used as a reference for both model inter-comparison and data/model comparison. X. Zhang et al.: LGM simulations and implications for deglaciation This hypothe- sis is supported by our LGM simulations and the hosing ex- periments, referring to its long-term equilibrium timescale from present-day ocean (about 5000 yr, even longer than the LGM) and distinct AMOC recovery processes to the FWP, respectively. In addition, model outputs from the simulation LGMS27ka further corroborate that the formation of AABW during 27 ka BP, as well as the AABW-cell are more ex- panded than during the LGM (Fig. 11), implying a critical role of the time interval, 27 ka BP, for the formation of the reconstructed LGM ocean structure (Duplessy et al., 1988; Curry and Oppo, 2005; Marchitto and Broecker, 2006) and the inception of the last deglaciation. Shown in Fig. 13 are the changes of sea temperature and salinity with time during the spin-up of LGM2PI and LGMS. In the spin-up of the LGM2PI, the upper layers of the ocean are warmed due to the warm boundary conditions (Fig. 13a), reducing the AMOC and NADW formation (Fig. S11). In this case the way that the bottom water mass interacts with the surface is mainly through the AABW formation in the Southern Ocean. In our climate model the major regions of AABW formation in PI are Antarctic on-shore areas where brine rejection occurs (Fig. 13a, b) due to sea ice formation and export. Given this, the warm upper-layer water mass can be transported to the bottom in the Southern Ocean (Fig. 13a) and destabilizes the ocean stratiﬁcation. Under LGM bound- ary conditions, the equatorward-extended permanent sea ice edge (Fig. 5) will shift the major AABW formation regions to the open ocean (Fig. 13c, d) where the dilution of the brines released by sea ice is more important and the effect of the brine-generated dense water is much more reduced than in on-shore regions (Bouttes et al., 2012). In addition, the cooled upper-layer water mass favors a strengthened AMOC during the spin-up of LGMS (Fig. S11), decelerating the northward extension of glacial AABW. Thus, the difference of the equilibrium timescale of deep ocean water mass be- tween PI and LGM boundary conditions can be attributed to the shift of AABW formation sites and different responses of the AMOC to the boundary conditions during the spin-up. 3.2 Potential origin of the glacial deep ocean Under the LGM (∼21 ka BP) boundary conditions the sim- ulated quasi-equilibrium ocean states starting from differ- ent initial ocean states in our climate model, i.e. LGMS- e and LGMW-e, are comparable to reconstructions (Dup- lessy et al., 1988; Curry and Oppo, 2005; Marchitto and Broecker, 2006; Lynch-Stieglitz et al., 2007) (Figs. 5–7 and S2), however, the equilibrium timescale in the simulations initialized from the glacial ocean is only about half of the simulation initialized from the present-day ocean state. Re- construction data indicates that δ13C-depleted and nutrient- rich water mass dominates the bottom of the Atlantic Ocean during the LGM, which is supposed to be a result from a Clim. Past, 9, 2319–2333, 2013 www.clim-past.net/9/2319/2013/ www.clim-past.net/9/2319/2013/ 2329 3.3 Differences of deep ocean equilibrium timescales between PI and LGM conditions In the following we investigate whether the dependence of equilibrium timescales on initial ocean states is also present in PI simulations. Figure 12 shows the meridional sections Clim. Past, 9, 2319–2333, 2013 www.clim-past.net/9/2319/2013/ (B) Implications for data/model and inter-model differences and last deglaciation: (B) Implications for data/model and inter-model differences and last deglaciation: Adkins, J. F., McIntyre, K., and Schrag, D. 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We thank the colleagues in Paleoclimate Dynamics group in the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research (AWI) in Bremerhaven for useful discussions. Thanks to Conor Purcell for help with English, Stephan Hageman in the Max Plank Institute in Hamburg for the HD model and the computer center of the AWI for the help in running the AWI supercomputer. Furthermore thank the reviewers and editor for constructive comments to improve the manuscript. X. Zhang was funded by China Scholarship Council (CSC). X. Zhang and G. Lohmann are funded through the PACES program of the AWI in the Helmholtz society in Germany. G. Knorr is funded by REKLIM. 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https://openalex.org/W2531610279	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0163537&type=printable	English	null	Fisher-Level Decision Making to Participate in Fisheries Improvement Projects (FIPs) for Yellowfin Tuna in the Philippines	PloS one	2,016	cc-by	12,198	RESEARCH ARTICLE OPEN ACCESS This study identifies the capabilities needed by small-scale fishers to participate in Fishery Improvement Projects (FIPs) for yellowfin tuna in the Philippines. The current literature pro- vides little empirical evidence on how different models, or types of FIPs, influence the par- ticipation of fishers in their programs and the degree which FIPs are able to foster improvements in fishing practices. To address this literature gap, two different FIPs are empirically analysed, each with different approaches for fostering improvement. The first is the non-governmental organisation-led Partnership Programme Towards Sustainable Tuna, which adopts a bottom-up or development oriented FIP model. The second is the pri- vate-led Artesmar FIP, which adopts a top-down or market-oriented FIP approach. The data were obtained from 350 fishers surveyed and were analysed using two separate mod- els run in succession, taking into consideration full, partial, and non-participation in the two FIPs. The results demonstrate that different types of capabilities are required in order to participate in different FIP models. Individual firm capabilities are more important for fishers participation in market-oriented FIPs, which use direct economic incentives to encourage improvements in fisher practices. Collective capabilities are more important for fishers to participate in development-oriented FIPs, which drive improvement by supporting fishers, fisher associations, and governments to move towards market requirements. Citation: Tolentino-Zondervan F, Berentsen P, Bush SR, Digal L, Oude Lansink A (2016) Fisher- Level Decision Making to Participate in Fisheries Improvement Projects (FIPs) for Yellowfin Tuna in the Philippines. PLoS ONE 11(10): e0163537. doi:10.1371/journal.pone.0163537 Editor: Jodie L. Rummer, James Cook University, AUSTRALIA Editor: Jodie L. Rummer, James Cook University, AUSTRALIA Received: November 24, 2015 Accepted: September 9, 2016 Published: October 12, 2016 Copyright: © 2016 Tolentino-Zondervan 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 research is funded by the Benefiting from Innovations in Sustainable and Equitable Tuna Management in the Coral Triangle and Western Pacific (BESTTuna) under the Interdisciplinary Research and Education Fund (INREF) programme of Wageningen University, The Netherlands. The BESTTuna website is found at http://www. wageningenur.nl/en/Research-Results/Projects- Frazen Tolentino-Zondervan1, Paul Berentsen1, Simon R. Bush2, Larry Digal3, Alfons Oude Lansink1 1 Business Economics Group, Wageningen University, Wageningen, The Netherlands, 2 Environmental Policy Group, Wageningen University, Wageningen, The Netherlands, 3 School of Management, University of the Philippines in Mindanao, Davao del Sur, Philippines, Tuna Value Chain Project, Commission on Higher Education, Davao City, Philippines a1111 frazen.tolentino@wur.nl * frazen.tolentino@wur.nl Fisher-Level Decision Making to Participate in Fisheries Improvement Projects (FIPs) for Yellowfin Tuna in the Philippines Frazen Tolentino-Zondervan1, Paul Berentsen1, Simon R. Bush2, Larry Digal3, Alfons Oude Lansink1 Fishers’ Decisions to Participate in FIPs designed to improve compliance with existing rules and management approaches [3–5]. The design and objectives of these private incentive mechanisms differ, but most commonly involve incentivising changes in fishing practices through value chain based arrangements such as industrial coalitions, improvement projects, and eco-certification(e.g. [6]). and-programmes/BESTTuna.htm. 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. The most dominant private incentive mechanisms for sustainable fisheries is the Marine Stewardship Council (MSC) standards, against which fisheries practices, the health of stocks and habitats, and the capacity of management to deliver sustainable outcomes are measured and certified[7]. However, one of the main criticisms of the MSC is its limited capacity to ade- quately include DCFs. As of 2015, only 8% MSC-certifiedfisheries are from developing coun- tries [8]. This limited inclusion is attributed to the high cost of certification,the lack of data on fish stocks available for assessment, and the inadequate or absence of effective governance and regulatory systems [9–12]. Recognisingthe difficultiesof DCFs to move towards certification,a range of Non-Government Organisations (NGOs) and private consultancy firms have devel- oped Fishery Improvement Projects (FIPs), a step-wise methodologyfor improving fisheries practices and management that originally started in developed world contexts but is also focused on DCFs [13–15]. FIPs utilize the market incentives in seafood value chains to stimulate sustainability improvements, which may or may not lead to MSC certification[15]. For example, retailers and food companies can push fishers towards improvements by directly funding a FIP or pur- chasing products (with or without a premium) from a fishery in a FIP [16]. The six stages FIP model proposed by the Sustainable Fisheries Partnership is as follows: 1. the identification of improvement goals and engagement of corporate partners; 2. agreement on work plans for improvement between fishers and participating partners; 3. engagement of regulators by FIP partners to improve regulation and market partners to adopt better product specification and procurement policies; 4. measurement of improvements to policy and practice; 5. key scientific indicators demonstrating a positive trend towards management goals; and 6. (optional) certifi- cation against the MSC standards [17]. Introduction The sustainability of fisheries is driven in large part by the alignment of fisher practices with management goals [1, 2]. State regulations, such as restrictions on fishing gears, harvest control rules, and access restrictions have traditionally been applied to change fisher behavior. How- ever, the perceived weakness of these state regulations, or total absence in many developing country fisheries (DCFs), has led to the emergence of private incentive mechanisms, which are PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 1 / 22 NE \| DOI:10.1371/journal.pone.0163537 October 12, 20 Fishers’ Decisions to Participate in FIPs (Thunnus albacares) in the Philippines. The first is the market-oriented Artesmar FIP, which is run by the private-company Meliomar and the consultancy firm BlueYou (Switzerland). This FIP sets a high sustainability requirement and provides economic incentives to encourage fish- ers participation. The second is the development-oriented Partnership Programme Towards Sustainable Tuna (PPTST) FIP, which is run by the World Wildlife Fund for Nature (WWF) Philippines, and seeks improved local governance of tuna fisheries to meet global value chain requirements. Both FIPs are focused on yellowfin tuna because of its market value and impor- tance to the local economy, the scalability of these FIPs to other sites in the Philippines and beyond targeting yellowfin tuna, and because yellowfin is a species subject to overfishing in recent years [22–24]. To understand the factors that influence fisher’s decisions to participate in FIPs, we employ a two stage framework including two models which are run separately. The following section describes both FIP models and the theoretical basis of capabilities and decision making. This is followed by an outline of the empirical data collection and of the probit and ordered probit models used for the two stage modelling. We then provide a justification for the variables adopted to explain fisher participation in FIPs. The paper concludes with a discussion of the key variables that are important for participation in different models of FIPs and recommenda- tions for enhancing fisher participation in FIPs. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 While differing in substance, other FIP models demon- strate a similar logic (see [18]) and are predicated on facilitating access to high end markets under the notional condition [15] that the fishery is working towards improvement. Despite convergence around the type of steps required, the mix of organisations involved, the kinds of fishers targeted, and the extent of institutional support provided in FIPs differ con- siderably. This is especially the case in the estimated 130-plus developing country FIPs [15]. Based on a recent attempt to create a general classification of FIP implementation [19] we define two general categories: ‘bottom-up’ development-oriented FIPs, often led by NGOs stimulating general improvements to government support and regulation; and ‘top-down’ market-oriented FIPs, led by firms focused on direct economic benefits for fishers in return for strict compliance [19]. It is assumed that these FIP models have consequences for the way fishers are included in FIP programs, especially in terms of the decision of fishers to change their practices in accor- dance with improvement criteria. Yet, there is little empirical evidence to verify this [14, 15]. We argue that the decisions made by fishers to participate in a FIP depends on the type of capabilities they have and whether these capabilities match with the requirements for participa- tion. These capabilities refer to the specific skills, practices, and forms of social organisations [20, 21]. This study classifies these capabilities into individual capabilities at personal level, individual capabilities at firm-level, and collective capabilities at the fishery or community level. By identifying these capabilities we build a clearer understanding of the specific factors that influence fisher decisions to comply with requirements that seek to improve fishing practices. The objective of this paper is to determine which decision making factors are important for small scale Filipino tuna fishers’ decisions to participate in two FIPs for yellowfin tuna 2 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs to prepare the fishery for MSC certification.Artesmar also sets chain of custody requirements, including strict quality standards for tuna, and traceability requirements such as vessel registra- tion and full catch documentation to verify the absence of Illegal Unreported Unregulated (IUU) fishing. Fishers participating in the Artesmar FIP are more likely to receive higher prices for their fish compared to fishers who do not participate because they are trained in how to handle their catch in such a way that improves the quality of the meat. They also have more certainty of having a buyer and receiving timely payment for the fish they land. These benefits of participation in the Artesmar FIP lower the risk of having a highly variable income, but are offset by the investment required for participation including time and effort allocated to train- ing, additional effort and investment to satisfy traceability requirements and upgrading facili- ties to meet fish safety and quality requirements. The cost of training is particularly high because of the structure of boat ownership, with fishers owning several boats having to extend new equipment, practices, and knowledge to their multiple boats and boat captains. The PPTST FIP is based on a public-private partnership established in 2011 to develop sus- tainable practices of yellowfin tuna fishers in Lagonoy and in Occidental Mindoro. Although funded by WWF Germany and European retailers such as Seafresh (Netherlands), Bell Seafood (Germany), and Coop (Switzerland), neither higher landing prices nor market access is cur- rently used as an incentive to participate in the FIP. Instead the PPTST FIP can be classified as a bottom-up comprehensive FIP, with day-to-day management of the FIP carried out by the WWF-Philippines and the municipal governments to improve the wider conditions of legal compliance and fisher safety in the fishery. Implementation of the PPTST FIP by WWF and the municipal governments focuses primarily on the organisation of fishers in associations, before engaging them in a consultative decision making process in order to comply with chain requirements similar to the Artesmar FIP (e.g. [25, 26]). The PPTST FIP targets a very wide range of fishers across the six municipalities of Sablayan, Mamburao, Paluan, Sta. Cruz, Calintaan, and Rizal, Occidental Mindoro (see Fig 1). Fishers in Sablayan and Mamburao are large scale and have adequate capital to meet requirements set by buyers in Metro Manila. Fishers in the other four municipalities have smaller scale operations, target species other than yellowfin tuna, and have other forms of livelihoods such as rice farm- ing, carpentry, and grocerystores. Unlike the Artesmar FIP which sets strict requirements for inclusion in the FIP, fishers can partially participate in the PPTST FIP by attending trainings related to fishery governance, without delivering tuna to the chain. Full participation requires the preparation of catch documents and providing export quality tuna based on their attendance at training sessions. In general, institutional support such as training, subsidies, and in-kind help are recognisedas important indirect incentives for the improvement of fisher practices (see [6]). Contrary to the trainings in the Artesmar FIP, the trainings in the PPTST FIP are organized and funded completely by external actors such as the WWF and the municipalities. The PPTST trainings are not only limited to complying with tuna quality and traceability requirements, but also extend to improving the governance of the fishery, such as putting in place anti-IUU mea- sures, and supporting the development of alternative livelihoods (such as ecotourism). Moreover these trainings reach different types of fishers ranging from small- to large-scale, including those fishers in remote areas of the municipalities. The trainings, subsidies, and in-kind help are con- sidered indirect incentives of the bottom up comprehensive FIPs because they are not directly associated with the market incentives of increased market access or a price premiums [6]. Participation in Different FIP Models Comparative FIP models In line with defining the different FIP models, the California Environmental Associates (CEA) [19] created four archetypes based on combination of two characteristic dimensions. The first dimension focuses on the structure of FIPs, ranging from ‘basic’ to ‘comprehensive’. Basic FIPs are characterised as a simple, low-cost model which provides small incremental improvements through time, while comprehensive FIPs are considered those that are resource intensive model and aimed at achieving MSC certification.The second dimension focuses on supply chain engagement along a spectrumof bottom-up vs. top-down. Bottom-up FIPs are those that develop improvements first and only later attempt to access high end markets and major buy- ers who have made sustainability commitments [19]. In contrast, top-down FIPs are those that start with the demand of major buyers and retailers to put pressure on fisheries to engage in sustainability in exchange for market access. The four possible archetypes from these two dimensions allow us to classify Artesmar as a top-down comprehensive FIP and PPTST as a bottom-up comprehensive FIP. Beginning in 2013, the goal of Artesmar is to provide market recognition and incentives for improved business and fishing practices of small scale fishers in the Philippines. Artesmar works in different regions of the Philippines, trading yellowfin from Occidental Mindoro, Albay, Quezon and Infanta, Antique, and Eastern Samar, Palawan, Batangas, Subic, Negros Occidental, and Zamboanga. Our research focused on Sablayan and Mamburao municipalities in Occidental Mindoro (see Fig 1) because the export chain for yellowfin tuna has been estab- lished since 2010 and full participation in the FIP is observable. Both of these municipalities are characterised by a higher concentration of fishers with larger tuna boats and higher tuna landings than the other municipalities of Occidental Mindoro. The two municipalities also have good access to tuna processing plants due to improved roads, proximity to landing sites for easier tuna transfer, and the availability of transportation and major servicessuch as com- munication and electricity which facilitates improved business transactions. The Artesmar FIP supports fishers to be compliant with legal catch documentation require- ments, as well as enhancing fishery data, and fishery co-management; all of which are necessary 3 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs Fig 1. The Occidental Mindoro fishery map. doi:10.1371/journal.pone.0163537.g001 Fig 1. The Occidental Mindoro fishery map. doi:10.1371/journal.pone.0163537.g001 doi:10.1371/journal.pone.0163537.g001 4 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ participation decision framework This study analyses fishers’ decisions to participate in the two FIPs (Fig 2). The first stage deci- sion concerns the choice to opt for participation in the Artesmar FIP or not. In principle, the 5 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs Artesmar FIP delivers higher returns and lowers the risk of fishers in terms of fluctuation in fish prices. However, higher investments for participation are required. The basic assumption here is that fishers who can fulfil the requirements of Artesmar FIP will choose this alternative because this option is expected to lead to a higher utility. As explained by the Utility Maximisa- tion (UM) framework, a rational individual will maximise his/her income and will minimise risks [27–29]. The second assumption is that participation in the Artesmar FIP, though pre- ferred by fishers, is not feasible for many fishers due to their lack of capabilities to comply with the requirements of the Artesmar FIP. Fishers that find themselves unable to participate in the Artesmar FIP might then opt for participation in the PPTST FIP as a second best option. Par- ticipation of a fisher in the PPTST FIP can be partial or full, depending on the fulfilment of requirements. As an extension of the utility framework, the third assumption is that the per- ceived social benefits of community membership (see [30–32]), adds to the utility of fishers when joining the PPTST FIP. The final option is that a fisher does not participate in any FIP. Testing the participation choices requires identifying significant variables that affect income and risks of fishers. The literature on decision making has extensively describedthe character- istics of decision makers that affect their level of income and risk (e.g. [33–36]). For this study, we divide these characteristics into four groups: individual capabilities at personal level, indi- vidual capabilities at firm-level, collective capabilities, and individual risk attitude and socio- demographic characteristics. Table 1 shows this grouping of characteristics, which are also used as the explanatory variables used to analyse decisions for FIP participation in the empiri- cal model explained below. Capabilities are defined as the specific skills, practices, and forms of social organisations required to deliver certain tasks in pursuit of long-term goals [20, 21]. The level of capabilities of individuals or firms influences the amount of risk they can handle and the income they can Fig 2. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs Table 1. The explanatory variables for fishers’ participation in Artesmar and PPTST FIPs. Characteristics of decision makers Deﬁnition of variables in Stata References FIP 1 if a ﬁsher belongs to non-Artesmar, 0 if a ﬁsher belongs to Artesmar Stages 1 if ﬁsher belongs to non-participation, 2 if partial participation in PPTST FIP, and 3 if full participation in PPTST FIP 1. Individual personal capabilities Fishing years Fishing experiences (in years) [34, 39, 47] Education Educational attainment of ﬁsher (1 if ﬁsher reaches high school, 0 otherwise) [34, 39, 47] 2. Individual ﬁrm capabilities Initial investment Initial investment for tuna ﬁshing boat (in ‘000,000 Philippine Pesos) [34, 76] Boat ownership 1 if tuna ﬁshing boat owner, 0 if non-boat owner [6] Boat capacity Tuna capacity of boats (in ‘000 kg) [6] Fishing trips Number of tuna ﬁshing trips in a month [42] Type of ﬁshing employment 1 if ﬁsher is ﬁshing tuna year round, 0 otherwise [42] Operating distance Tuna ﬁshing operating distance (in km) [42] Fishing days Number of tuna ﬁshing days [42] 3. Collective capabilities Membership to an association 1 if ﬁsher has membership to a ﬁsher association, 0 otherwise Interviews, [6] Trainings and subsidies 1 if a ﬁsher receives trainings and subsidies from government, 0 otherwise Interviews, [6] Financing operation 1 if ﬁsher personally ﬁnances his ﬁshing operation, 0 if ﬁnances by Casas Interviews, [6, 43, 44, 50] 4. Individual risk attitude and socio demographic Risk Attitude 1 if ﬁsher sells in quality method, 0 if straight method [34, 35, 46] Age Individual ﬁsher’s age (in years) [34, 47] Number of family members Number of immediate family members [34] Other source of income 1 if ﬁsher has other sources of income, 0 otherwise [48, 49] doi:10.1371/journal.pone.0163537.t001 generate [37]. For instance, a fisher that is more capable of adopting a certain fishing technol- ogy will find the risk of adoption lower than a fisher who still needs to build his/her capability to adopt the fishing technology. Moreover, the adoption of a fishing technology by a more capable fisher increases the likelihoodof gaining a higher income [34, 35, 38]. The capabilities are expressed at the individual, firm and collective levels. Fishers’ participation decision framework The decision model for fisher participation. doi:10.1371/journal.pone.0163537.g002 Fishers’ Decisions to Participate in FIPs Fig 2. The decision model for fisher participation. Fig 2. The decision model for fisher participation. Fig 2. The decision model for fisher participation. doi:10.1371/journal.pone.0163537.g002 doi:10.1371/journal.pone.0163537.g002 Artesmar FIP delivers higher returns and lowers the risk of fishers in terms of fluctuation in fish prices. However, higher investments for participation are required. The basic assumption here is that fishers who can fulfil the requirements of Artesmar FIP will choose this alternative because this option is expected to lead to a higher utility. As explained by the Utility Maximisa- tion (UM) framework, a rational individual will maximise his/her income and will minimise risks [27–29]. The second assumption is that participation in the Artesmar FIP, though pre- ferred by fishers, is not feasible for many fishers due to their lack of capabilities to comply with the requirements of the Artesmar FIP. Fishers that find themselves unable to participate in the Artesmar FIP might then opt for participation in the PPTST FIP as a second best option. Par- ticipation of a fisher in the PPTST FIP can be partial or full, depending on the fulfilment of requirements. As an extension of the utility framework, the third assumption is that the per- ceived social benefits of community membership (see [30–32]), adds to the utility of fishers when joining the PPTST FIP. The final option is that a fisher does not participate in any FIP. Testing the participation choices requires identifying significant variables that affect income and risks of fishers. The literature on decision making has extensively describedthe character- istics of decision makers that affect their level of income and risk (e.g. [33–36]). For this study, we divide these characteristics into four groups: individual capabilities at personal level, indi- vidual capabilities at firm-level, collective capabilities, and individual risk attitude and socio- demographic characteristics. Table 1 shows this grouping of characteristics, which are also used as the explanatory variables used to analyse decisions for FIP participation in the empiri- cal model explained below. Capabilities are defined as the specific skills, practices, and forms of social organisations required to deliver certain tasks in pursuit of long-term goals [20, 21]. The level of capabilities of individuals or firms influences the amount of risk they can handle and the income they can 6 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs market requirements the Artesmar FIP demands a higher level of capabilities for participation. This study hypothesizes that individual capabilities at firm-level will increase the likelihoodof fisher participation to the Artesmar FIP because these variables reflect the higher capabilities of fishers. The individual risk attitude and socio-demographicvariables influence the required income and acceptable risk for the decision maker [34, 35]. This group of variables includes risk atti- tude, age, number of family members, and other sources of income (Table 1). The risk attitude reflects the extent to which decision makers value risks [46]. A value of 1 in the variable ‘risk attitude’ (Table 1) corresponds to the risk fishers take on by having a preference for what is termed locally as ‘quality buying’—a method that requires quality inspection of tuna and assigns higher price for export quality tuna while lower price for rejected quality tuna. In this study, fishers with lower risk aversion (i.e. risk takers) will more likely choose the alternative that will give a higher expected income [33, 34, 38]. This study assumes that socio-demo- graphic variables may result in two opposite effects, which can influence the significanceof these variables in the model. For instance, the age of the fisher affects the time horizon with which an investment can be recovered [34, 47]. A higher age is therefore expected to decrease the likelihood for participation in the Artesmar FIP. At the same time, a higher age may also mean fishers have more fishing experience to improve their compliance with market require- ments and could therefore increase the likelihoodof participation in the Artesmar FIP [34]. Similarly a higher number of family members may reduce the risk of a fisher or firm by increas- ing the availability of labour, thus increase the likelihoodof participation in the Artesmar FIP [34]. However, the number of family members also places greater pressure to remain economi- cally viable and food secure, which can lead to a lower probability of participation in the Artes- mar FIP. Additional sources of income may reduce this risk by providing alternative sources of finance to firms, which in turn may increase the funds available and the likelihood of participa- tion in the Artesmar FIP [48]. As shown in Table 1, individual capabilities of the fishers correspond to what others have termed human capital, including the experiences and education of an individual, and can be used as a proxy for the competences and skills either inherent or acquired by that individual [39]. Firm capabil- ities refer to the collection of competences of the organisation to which the individual belongs [39–41] and reflect the use of material resources in order to comply with transaction require- ments [6]. In this study boats are considered as the unit of a firm operated by individuals. The material resources that are translated to individual firm capabilities include initial investments, boat ownership, boat capacity, type of fishing employment, number of fishing trips, operating distance, and fishing days [6]. These last three factors represent the input variables used by fishers, including gasoline/fuel,ice, and labor costs [42]. Collective capabilities correspond to what others refer to as social capital, including shared resources acquired through external rela- tionships and networks that improve personal- and firm-level capabilities [39]. In practical terms this extends to membership to a fisher association, institutional support such as trainings and subsidies, and financing business operations through loans either from an association or from Casas–local elites or patrons who control trade and provide credit for fishing, as well as for household needs such as education and health (see [43–45]). Based on compliance with 7 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Total number of surveys carried out is 350. However, the number is reduced to 316 due to incomplete and non-responses of some ﬁshers. Fishers’ Decisions to Participate in FIPs markets to fishers for selling their fish; and by 2) the lack of trusts of fishers in those assessing quality at landing sites [50]; (3) the increasing temperature that results to low tuna quality; and (4) the decreasing tuna catches in the recent years that causes fluctuation in income [51]. Based on the assumptions outlined here, the rest of the paper addresses two key questions. First, which variables are important for fishers participation in the Artesmar and PPTST FIPs? Second, based on these variables, to what extent do top down and bottom up comprehensive FIPs facilitate participation of fishers in their programs? markets to fishers for selling their fish; and by 2) the lack of trusts of fishers in those assessing quality at landing sites [50]; (3) the increasing temperature that results to low tuna quality; and (4) the decreasing tuna catches in the recent years that causes fluctuation in income [51]. Based on the assumptions outlined here, the rest of the paper addresses two key questions. First, which variables are important for fishers participation in the Artesmar and PPTST FIPs? Second, based on these variables, to what extent do top down and bottom up comprehensive FIPs facilitate participation of fishers in their programs? doi:10.1371/journal.pone.0163537.t002 Source: WWF Philippines, June 2014 At the same time, other sources of income are also assumed to increase risk due to the allocation of time and resources to other activities, thus decreasing the likelihoodof participation in the Artesmar FIP [49]. In the second-stage decision, collective capabilities such as membership to an association, and subsidies from the government enable fishers to comply with the requirements of the development-oriented FIP, while the financial dependencyon Casas is either improving or lim- iting the participation of fishers. These collective capabilities reduce the exposure of fishers to risks by acquiring parts of investments to external actors and developing fishers’ capabilities through bottom-up comprehensive FIPs, and thus improve their income. We assume that membership to a fisher association strengthens the social relationships among fishers, enables fishers to improve the management of their fisheries, and allows access to the external resources that help fishers to improve their fishing activities in order to comply with FIP requirements [6]. Fishers, therefore, will perceive higher utility in joining a bottom-up compre- hensive FIP given their focus on establishing fisher associations. Finally, we assume that the financial dependency of fishers to Casas can motivate fishers to participate in these FIPs because of (1) fishers feelings of indebtedness to Casas and/or (2) the (mis)trust of Casas that prices are correlated with the quality of fish. At the same time, the financial dependency of fish- ers to Casas may limit their freedom in joining a FIP in which they are not involved, because Casas may lose control over fishers which is based on a mix of familial and debt-tied relations [43, 44, 50]. The non-participation of fishers in all FIPs may not only be attributable to a lack of capabili- ties only, but also to a higher perceived risk of participating in a FIP. Field results indicate that the risk perception of fishers is mainly influenced by four factors: 1) the existing tied-credit relation of fishers to Casas that results to lower fish price, while leaving no alternative higher 8 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Source: WWF Philippines, June 2014 Data Collection Individual fisher data were obtained from a survey covering the six municipalities of Occiden- tal Mindoro. The Wageningen School of Social Sciences (WASS) at Wageningen University assessed and approved the research proposal in 2013 before fieldwork commenced in 2014. Verbal consent was sought from fishers by first explaining to them the objective of this study, the kind of information this study requires, and the use of the information they will provide. The interviews commenced after verbal consent was given and recorded by including the name of the fishers in the survey form. Written consent was not obtained due to the illiteracy of most of the fishers and the sensitivity of some fishers in terms of signing any form of what they per- ceive to be formal documentation containing their names. The total fisher population in Occidental Mindoro is 3584 fishers, according to the database of WWF Philippines as of June 2014. Following Field [52], a total of 350 randomly selected sur- veys were carried out with the target of 10% of the population across spatially dispersed areas per municipality (see Table 2). The number of complete responses was 316, which can be divided into four groups according to their participation in the Artesmar FIP (6.3%), full par- ticipation in the PPTST FIP (17.4%), partial participation in the PPTST FIP (34.2%), and non- participation (42.1%) (see Table 3). The number of Artesmar FIP fishers is partly limited since the Artesmar FIP started two years later than the PPTST FIP and only covered two out of six FIP municipalities in Occidental Mindoro. Table 3 also shows descriptive statistics of the independent variables that were used to explain fisher participation. In general, the scale variables such as fishing years, initial invest- ments, boat capacity, operating distance, number of fish days, age, and number of family mem- bers show an increasing trend with increasing levels of participation, while the number of fishing trips shows a decreasing trend. The distribution of fishers is also shown for each nomi- nal variable. The education level of fishers is evenly distributed across four fisher participation Table 2. The distribution of samples in each municipality. Municipality Total population** Number of samples* Sablayan 1656 165 Mamburao 850 85 Paluan 113 12 Sta. Cruz 350 25 Calintaan 315 33 Rizal 300 30 Total 3584 350 Table 2. The distribution of samples in each municipality. Table 2. Data Collection The distribution of samples in each municipality. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 9 / 22 Fishers’ Decisions to Participate in FIPs Table 3. Descriptive statistics of variables based on four groups of fishers participation in FIP. Variables Artesmar FIP (n = 20) 6.3% Full PPTST FIP (n = 55) 17.4% Partial PPTST FIP (n = 108) 34.2% No Participation (n = 133) 42.1% Scale variables Fishing years 23.95 (2.7410) 21.50 (1.6487) 19.03 (1.09) 18.81 (0.9835) Initial investments 3.79 (2.2136) 1.98 (0.3295) 0.61(0.1090) 0.74 (0.1423) Boat capacity 1.74 (0.3172) 1.25 (0.1280) 0.90 (0.1575) 0.90 (0.0948) Number of ﬁshing trips 3.9 (1.0709) 3.60 (0.3349) 8.96 (0.8252) 7.67 (0.7033) Operating distance 46.25 (9.9331) 50.27 (5.3346) 35.10 (2.8944) 35.54 (2.6733) Number of ﬁsh days 6.6 (0.6257) 6.05 (0.3571) 4.15 (0.2737) 4.69 (0.2309) Age 44.75 (2.8533) 42.45 (1.3530) 41.81 (1.1230) 40.53 (0.9858) Number of family members 6.25 (0.5020) 4.96 (0.2854) 5.35 (0.2245) 5.16 (0.1801) Nominal variables Frequency count (%) Frequency count (%) Frequency count (%) Frequency count (%) Education Reach Hs (1) 10 (50) 55 (50.9) 57 (42.9) 30 (54.5) Do not reach Hs (0) 10 (50) 53 (49.1) 76 (57.1) 25 (45.5) Membership to an association Yes (1) 4 (20) 67 (62.0) 12 (9.0) 44 (80.0) No (0) 16 (80) 41 (38.0) 121 (90.10) 11 (20.0) Trainings and subsidies Yes (1) 3 (0.15) 55 (50.9) 17 (12.8) 22 (40.0) No (0) 17 (0.85) 53 (49.1) 116 (87.2) 33 (60.0) Boat ownership Boat owner (1) 8 (40) 78 (72.2) 71 (53.4) 42 (76.4) Not owner (0) 12 (60) 30 (27.8) 62 (46.6) 13 (23.6) Financing operation Personal (1) 8 (0.4) 61 (56.5) 70 (52.6) 18 (32.7) Casa (0) 12 (0.6) 47 (43.5) 63 (47.4) 37 (67.3) Season Full-time (1) 8 (40) 34 (31.5) 51 (38.3) 46 (83.6) Part-time (0) 12 (60) 74 (68.5) 82 (61.7) 9 (16.4) Risk attitude Quality (1) buying 9 (0.45) 13 (12.0) 11 (8.3) 18 (32.7) Straight (0) buying 11 (0.55) 95 (88.0) 122 (91.7) 37 (67.3) Source of income Yes (1) 10 (50) 78 (72.2) 67 (50.4) 25 (45.5) No (0) 10 (50) 30 (27.8) 66 (49.6) 30 (54.5) doi:10.1371/journal.pone.0163537.t003 Table 3. Descriptive statistics of variables based on four groups of fishers participation in FIP. options. The membership to a fisher association and the boat ownership of fishers increase from non-participation to full participation in the PPTST FIP. Artesmar FIP has the highest fraction of risk taking fishers. Also the percentage of fishers that prefers quality buying increases with the degree of participation in the PPTST FIP. options. The membership to a fisher association and the boat ownership of fishers increase from non-participation to full participation in the PPTST FIP. Artesmar FIP has the highest fraction of risk taking fishers. Also the percentage of fishers that prefers quality buying increases with the degree of participation in the PPTST FIP. options. The membership to a fisher association and the boat ownership of fishers increase from non-participation to full participation in the PPTST FIP. Artesmar FIP has the highest fraction of risk taking fishers. Also the percentage of fishers that prefers quality buying increases with the degree of participation in the PPTST FIP. The sample selection equation is represented by: z ¼ a’w þ u z ¼ 1½z > 0 The variable z is a shadow variable ruling the fisher participation in Artesmar FIP, α0 is a coefficientof the selection process, w is the explanatory variables known to influence the selec- tion decision, and μ is the random error term of sample selection equation that is normally dis- tributed. When z = 1, the fisher belongs to the category ‘non-Artesmar’ (and z = 0 for the Artesmar FIP) since further analysis will be done for a group of fishers not involved in the Artesmar FIP. All variables in Table 1 are used in the selection equation. Next is to use the result in the first equation to calculate the inverse Mills ratio, a selection hazard that is added as an explanatory variable in the second equation to remove the sample selection bias. Similar to Heckman’s two-stage sample selection model and to other studies (e.g. [59–63]) that employed the same methodology, the second stage is an ordered probit equation. Since the second stage applies for the six regions where PPTST FIP operates (exclud- ing Artesmar FIP in the analysis), individual fishers in Mamburao and Sablayan have values in their inverse Mills ratio while fishers in the remaining four municipalities have inverse mills ratio set to zero. The ordered probit equation is: y ¼ b0x þ g l þ ε; y ¼ j if mij < y mj: y ¼ j if mij < y mj: The y represents a shadow variable ruling the ordered stages of participation to which non- Artesmar fishers belong: β’ represents the coefficientof outcome explanatory variables x, γ is the selectivity bias, λ is the inverse Mills ratio, and ε is the random error term for the ordered probit equation. The y = j corresponds to the exact stage of participation in non-Artesmar, and is decomposed as follows: 1(y = 1) for non-participation, 2 for partial participation in the PPTST FIP, and 3 for full participation in the PPTST FIP. The variables in Table 1 are used in the ordered probit equation. Before running the probit and the ordered probit models, two steps were conducted. First, all independent variables were checked for multicollinearity using the Variance Inflation Factor (VIF), which in this case gave values well below 10 for all explanatory variables (see S2 Table under Supporting Information). The sample selection equation is represented by: This means that all explanatory variables exhibit low multicol- linearity so all variables are retained in the model. Second, the heteroskedasticity of the model was tested using the Breusch-Pagan test to determine whether the variance of the error terms increases or decreases with explanatory variables. The heteroskedasticity test shows a signifi- cant result (see S3 Table), implying that running the model using robust standard errors reduces the variance of the error terms. Finally we added two extra steps to the model to test the robustness of our result, as shown in the supplementary information (see S5 and S6 Tables). First, we checked for the significance of the inverse Mills ratio using the samples in Mamburao and Sablayan. Second, we ran a sepa- rate ordered probit model in the second stage that does not account for inverse Mills ratio for comparison with the one that does account for inverse Mills ratio. The empirical model In modelling fishers’ decision making to participate in FIPs, fishers decisions are structured in the two stages as explained above (Fig 2). The initial explanatory variables to explain fisher’s decision making are outlined in Table 1 and are coded in Stata 13, the statistical program used in this paper. We employed a two-stage modelling approach, similar to the concept of Heckman’s two- stage sample selection model [53–56], because it enables us to assess fisher’s participation in FIPs in two stages [57, 58]. The first stage is a sample selection equation that uses a probit model. In this study, the sample selection deals with fishers options to choose between Artes- mar FIP or non-Artesmar, which works for the two municipalities of Sablayan and Mamburao. 10 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs The sample selection equation is represented by: Fishers’ Decisions to Participate in FIPs PPTST FIP and the ordered outcome of non-participation in FIP, partial and full-participation in PPTST FIP. 0.01 esmar FIP and 0 = non-Artesmar i li i f S bl d M b Th l b f i 222 b S li i b i i h esmar FIP and 0 = non Artesmar cipalities of Sablayan and Mamburao. The actual number of surveys is 222, but Stata eliminates observation with aFor reading the result, let 1 = Artesmar FIP and 0 = non-Artesmar Note: The ﬁsher data involve municipalities of Sablayan and Mamburao. The actual number of surveys is 222, but Stata eliminates observation with incomplete responses Participation in Artesmar FIP Table 4 shows all independent variables that might explain participation of fishers in the Artes- mar FIP, together with the corresponding coefficients,standard error, and Z-value. The coeffi- cients are interpreted based on the direction of the effect of the variables [56]. For instance, a positive coefficientmeans that a variable increases the participation of fishers in Artesmar FIP while a negative coefficientreduces the participation of fishers in Artesmar FIP. It is not possi- ble to use the value of these coefficientsin estimating the increase or decrease of participation because the coefficientsare not derived using a linear model. The inverse Mills ratio shows insignificant result which means that there is no evidence that selection bias is quantitatively important. The results presented in Table 4 show that some explanatory variables have significant effects on fishers’ choice for Artesmar FIP over non-Artesmar. The fit of the model based on the probit equation is P > χ2 = 0.00, which implies a good model fit at α = 0.01. Consistent Table 4. Results of the probit model in the first stage using data from Sablayan and Mamburao fishers. Coefﬁcients Standard Error Z FIPa Individual personal capabilities Fishing years 0.005 0.014 -0.35 Education 0.156 0.303 -0.51 Individual ﬁrm capabilities Initial investment 0.089* 0.029 -3.082 Boat ownership -0.391 0.351 1.11 Boat capacity 0.035 0.085 -0.42 Fishing trips 0.011 0.044 -0.25 Type of ﬁshing employment -0.836* 0.300 2.78 Operating distance -0.003 0.005 0.53 Fishing days 0.070 0.053 -1.32 Collective capabilities Membership to Association -0.269 0.330 0.82 Financing operation -0.276 0.279 0.99 Trainings and subsidies -0.549 0.414 1.33 Individual risk attitude and socio demographic Risk attitude 1.132* 0.293 -3.86 Age 0.017 0.018 -0.98 Number of family members 0.259* 0.069 -3.78 Other sources of income 0.119 0.279 -0.42 constant 3.80 *** 0.941 4.03 N = 220; Wald chi(16) = 47.60; prob>(chi)2 = 0.000; inverse mills ratio = 0.16 * Statistical signiﬁcance at α = 0.10 Statistical signiﬁcance at α = 0.05 * Statistical signiﬁcance at α = 0.01 aFor reading the result, let 1 = Artesmar FIP and 0 = non-Artesmar Note: The ﬁsher data involve municipalities of Sablayan and Mamburao. The actual number of surveys is 222, but Stata eliminates observation with incomplete responses. doi:10.1371/journal.pone.0163537.t004 4. Results of the probit model in the first stage using data from Sablayan and Mamburao fishers. Table 4. Results First the modelling results will be presented for the first stage decision on participation in the Artesmar FIP, followed by the results for the second stage decision on participation in the 11 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs with our hypothesis, the initial investment for fishing boats, risk attitude, and number of family members are significant and positively contribute to the fishers’ choice to participate in the Artesmar FIP. Increasing initial investment for fishing boat reflects higher capabilities of fish- ers in terms of boat size, boat engine, and fishing equipment. These capabilities enable fishers to deliver tuna in the chain, therefore increasing their likelihoodof choosing the Artesmar FIP and their chances of gaining higher income to recover their investments. A higher value for risk attitude of fishers as manifested by fishers preference to sell through quality buying, increases the likelihoodof fishers to participate in the Artesmar FIP. A higher number of family members indicates that fishers also have an increase likelihood to participate in the Artesmar FIP. Based on field observations and interviews,fishers participating in Artesmar FIP often operate as part of a large scale family business. As shown in the literature [64], family members in the fishing business reduce fishers’ risks by having trusted employees, by developing the capabilities of the family members in order to succeed the business in the future, and by having confidence that a fisher contributes to the welfare of the family member. Contrary to what was expected, the type of fishing employment has a negative effect on par- ticipation in the Artesmar FIP. The results show that the more fishers operate part-time or tar- get species aside from tuna, the more likely it is that they will participate in the Artesmar FIP. Most part-time tuna fishers shift from tuna fishing to catching other species during the low sea- son for tuna catches in order to recover their costs of operation. Moreover, based on field inter- views, some fishers believe that tuna stocks are declining over time, and therefore search for alternative catch to balance the risks of going out to sea. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Participation in Artesmar FIP Results of the probit model in the first stage using data from Sablayan and Mamburao fishers. C S Z PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 12 / 22 Participation in PPTST FIP The results of the ordered probit model on the degree of participation of fishers in the PPTST FIP are presented in Table 5. Variables such as type of fishing employment, operating dis- tance, membership to an association, and risk attitude have positive and significant effects on the degree of fishers’ participation. The type of fishing employment increases the likelihood of full-participation in PPTST FIP when fishers target tuna on a full-time basis. Fishers spe- cializing in tuna may have better skills in catching and improving tuna as compared to diver- sified fishers, thus enabling them to comply with the product requirements of PPTST FIP and therefore increasing their chance of generating higher income. As the operating distance increases, the likelihood of full participation in the PPTST FIP also increases. Fishers also note that the declining catch in municipal water (<15 km from seashore) drives them further seaward to search for better fishing grounds. Doing so increases their chances of catching and delivering tuna in the PPTST FIP, which in turn increase their chances of gaining higher income. The membership to a fisher association also increases the likelihood of fishers to move from non-participation to full-participationin PPTST FIP. Based on interviews,fishers in the PPTST FIP perceive that joining a fisher association will help them improve the quality of their tuna, and will finally lead to higher tuna prices. The fisher association increases the perceived benefits of fishers since the association helps fishers to generate funding and subsidy such as fishing aids from the government, which improves their fishing activities. Moreover, a fisher association also organises activities such as the marine guards to prevent illegal fishing and conservation activities as part of the improvement process in the FIP. Finally, the risk attitude of fishers shows a positive effect in terms of pushing fishers to fully participate in the PPTST FIP. This means that fishers are willing to sell more through quality buying because these fish- ers are confident that they acquired the knowledge and skills to improve the quality of tuna catches, which in turn increases the probability of getting higher prices for the fish they land. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 13 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs Fig 3. The marginal effects of significant variables in second stage decision after ordered probit model. Note: The numbers in bold represent the marginal effects significant at α = 0.05. Fig 3. The marginal effects of significant variables in second stage decision after ordered probit model. Note: The numbers in bold represent the marginal effects significant at α = 0.05. doi:10.1371/journal.pone.0163537.g003 doi:10.1371/journal.pone.0163537.g003 membership to a fisher association as an example, the graph shows that when any fisher is a member of a fisher association, the probability of not participating reduces by 51.5%, while the probability of partial participation increases by 21.4%, and of full participation increases by 22.7%. In another example, an addition of one km to the 35.5 km average operational distance (see Table 3) will reduce the non-participation of fishers by 0.2% (see Fig 3). Aside from the direct interpretation of the marginal effects, the graph also shows the trends of marginal effects in selecting for partial or full participation in the PPTST FIP. The five variables show an increasing trend, which means that they generally enhance the participation of fishers from non- to full-participationin the PPTST FIP. The variables membership to a fisher associa- tion and risk attitude are highly significant in non, partial, and full-participationin PPTST FIP at α = 0.01. This means that both variables are highly important in improving the participation of fishers in the PPTST FIP. The type of fishing employment, operating distance, and financing operation are significant at α = 0.05 and at 0.10. It is also notable that among the five variables, the membership to a fisher association shows the highest marginal values, making it highly important for both partial and full participation. The majority of the partial participants are able to get funding from the government to improve their livelihoods through a fisher association. Full participants also benefit by being members of a fisher association by having regulatory sup- port to fight IUU fishing in their area, thus ensuring greater legality of fish caught and traded. Fishers’ Decisions to Participate in FIPs Table 5. Results of the estimation of the ordered probit model in six municipalities of Occidental Mindoro. Coefﬁcients Standard Error Z Stagesa Individual personal capabilities Fishing years 0.012 0.007 1.52 Education 0.089 0.152 0.58 Individual ﬁrm capabilities Initial investment 0.074 0.061 1.21 Boat ownership 0.227 0.208 1.09 Boat capacity -0.037 0.071 -0.53 Fishing trips -0.009 0.014 -0.65 Type of ﬁshing employment 0.407 0.188 2.16 Operating distance 0.005 0.003 2.03 Fishing days 0.026 0.040 0.65 Collective capabilities Membership to Association 1.484* 0.185 8.01 Financing operation -0.241 0.159 -1.52 Trainings and subsidies 0.078 0.196 0.4 Individual risk attitude and socio demographic Risk attitude 0.770* 0.254 3.03 Age 0.003 0.008 0.41 Number of family members -0.034 0.038 -0.88 Other sources of income -0.304* 0.157 -1.93 Inversemills1 -1.083 0.771 -1.4 Cut1 1.04 0.455 Cut2 2.57 0.474 Z Having alternative sources of income has a negative and significant effect on the participa- tion of fishers in the PPTST FIP as compared to those fishers who solely rely on fishing as a source of income. Based on interviews,and consistent with literature related to fishing as a form of employment (e.g [49]), some fishers consider fishing as the poorest among other forms of employment and would like to move away from fishing if possible. Therefore, having other sources of income such as farming and small enterprises leads them to prioritize alternative sources of income rather than improving their fishing practices to satisfy FIP requirements. Finally, the significant variables in the ordered probit model are further analysed by deriving their marginal effects, which show the change in probability when an independent variable increases by one unit. Calculating marginal effects in the second stage decision shows both the patterns and requirements for fishers to move from one participation stage to another. Fig 3 shows these marginal effects of the type of fishing employment, operating distance member- ship to an association, risk attitude, and other sources of income based on non-, partial-, and full-participationin the PPTST FIP (also refer to S3 Table for detailed information). The verti- cal axis of the graph represents the values of marginal effects from low to high, while the data labels or numbers in bold represent the significance of marginal effects at α = 0.05. Taking the PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 14 / 22 Capabilities, risk and FIP participation The results show that different types of capabilities influence the decision of fishers to join dif- ferent kinds of FIPs. First, individual firm capabilities, such as initial investment and type of fishing employment, increase the likelihoodfor fisher participation in response to market-ori- ented FIPs, which provide economic incentives through dock side transactions based on qual- ity. These individual firm capabilities are more likely to be exhibited by those with considerable financial capacity, owning and financing several fishing boats. Moreover, these fishers are also more likely to be engaged in other chain-related activities. However, consistent with now wider observations of how economic decisions are embedded in wider social or community relations [65, 66], the results also show that these individual firm capabilities are not independent from wider social relations in which these firm actors find themselves in fishing communities. For example, socio-demographicvariables, such as the higher number of family members, are also shown to play a role in the probability of participating in market-based FIPs possibly because familial ties reduce the risk associated with individual choices to invest in complying with FIP requirements [64, 67]. Second, collective capabilities, such as membership to an association, are more likely to increase participation in the bottom-up PPTST FIP. The results also show that full participa- tion in development-oriented FIPs remains dependent on the characteristics of the individual fishing firm. The results indicate that fishers fully participating in PPTST FIP have a greater degree of operation, for instance by their large capacity to finance high costs of operation at a farther distance and through their capability to cope with risks associated with full-time tuna fishing particularly from March to September where catches are lower (see for example [68]). However, partial participation is dominated by those fishers with smaller ‘one-man, one-boat’ operations, characterised by fishing at shorter distance from shore, and who are dependent on collective capabilities supported by fishing associations and Casas (see [69] for comparable results). The consequence appears to be that despite the bottom-up oriented nature of the PPTST FIP, targeting a wider group of smaller scale and relatively poorer fishers, full participa- tion appears to remain linked to those with higher individual firm level capabilities. This sup- ports the idea of “elite capture” of benefits [70], meaning that fishers with a high level of individual capabilities, in general, benefit from participation in FIPs. Fishers’ Decisions to Participate in FIPs comprehensive FIPs, and on the influence these factors have on the design of different FIPs as a means of reaching goals of improved fishery practices and fisheries management. Capabilities, risk and FIP participation In addition to capabilities, the results indicate that risk attitude also influences the decision of fishers to participate in the two FIPs. First, the results indicate that less risk averse fishers are more likely to participate in the top-down Artesmar FIP. Building on work around fisher behaviour in response to economic risk [39, 40, 46], this finding indicates that those fishers choosing for Artesmar FIP do so in order to not only generate higher income, but more impor- tantly to reduce the lack of transparency and high price volatility inherent in the landings sites controlled by Casas. This means that the high price in Artesmar FIP reduces the risks of fishers against lower fish prices, as long as these fishers satisfy the fish quality requirement. In contrast, those choosing for the bottom-up PPTST FIP were not able to reduce this risk, as seen in many small-scale developing country fisheries [71, 72], because most buyers involved were not mov- ing to quality buying and maintained many of the same debt-tied relations. Discussion Based on these results we now further elaborate on how the capabilities of fishers influences fisher decision making for different levels of participation in top-down and bottom-up 15 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs environmental improvement (such as eco-certification,see [73]), top down comprehensive FIPs requiring high level of individual capabilities have a higher risk of being selective of those fishers who can more easily comply with sustainability standards in exchange for immediate market access [6]—with the consequence of reducing the potential for overall fishery improve- ment. In contrast, bottom up comprehensive FIPs fostering collective capabilities appear to be more inclusive of a larger group of fishers, but with the potential consequence of delivering lower overall improvements to the fishery as a whole. Balancing these trade-offs is at the core of the differentiation on how different FIP models are evolving [19]. Second, the results indicate that the design of FIPs also influences the delivery of incentives to fishers, which also affects inclusiveness and overall impact. As argued above, top-down FIPs appear to be better able to deliver more transparent price signals to fishers. The case of the Artesmar FIP shows that companies like Meliomar invest in vertical coordination and in doing so control the chain from production to export. They are thus better able to set higher prices for higher quality fish and to transmit this price incentive to fishers [74, 75]. On the other hand, bottom-up FIPs with weaker relationships between retailers and fishers may result in weaker control over chain coordination. In addition, the on-ground implementation by actors operating outside the chain such as WWF, appears to result in weaker delivery of price-related incentives. Based on field interviews and observation,WWF serves as a facilitator outside the chain that links fishers to other seafood stakeholders. However, WWF does not have influence over the price of fish. Nevertheless, supporting other observations [6, 14], bottom-up compre- hensive FIPs do appear to offer more durable support from government given their closer rela- tionship during implementation. Finally, these results open up questions around the degree of complementarity and competi- tion that might exist between FIP models in relation to inclusion and improvement. While the results point to the differences between top-down or bottom-up comprehensive FIPs, it may not necessarily be the case that these or other FIP models are opposed or in competition. Instead, building on nascent ideas of FIP design [19], two FIP models operating in one place points to a range of potential complementarities in terms of delivering incentives to fishers and improving the governance of the fishery. Here the more stringent top-down market-oriented approach could target fishers with individual firm capabilities, while bottom up ‘development- oriented’ FIPs could provide a more inclusive approach. By targeting different capabilities, and setting different incentives, the two models could work in tandem to provide overall greater gains in fishery improvement. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Consequence for FIP design Our results hold consequences for the design of FIPs, as well as the relationship between differ- ent FIP models. First, reflecting wider discussions on market-based approaches to 16 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 Fishers’ Decisions to Participate in FIPs We also conclude that in order to increase the participation of fishers, those designing FIPs need to not only recognize the economic utility that a fisher could derive from participation, but also to identify and build fishers capabilities to participate in a FIP. Moreover, FIPs must also balance both the deliverance of incentives to individual fishers and the improvement in the governance of the fishery in order to enhance participation of fishers and thus achieve high sustainability impact. Future research may elaborate on the implementation of FIPs to poor and well performing fishers and to different fisheries (e.g. tuna or other species), and on their consequences to the inclusiveness and fishery improvement. Finally, focusing on ways to enhance the participation of fishers in FIPs and in other private incentive mechanisms is also relevant to achieve higher sustainability outcomes. Supporting Information S1 File. Estimation of the sample size. (PDF) S2 File. Survey forms on household fishers. (PDF) S1 Table. Data of individual fisher surveysin Occidental Mindoro fishery. (XLSX) S2 Table. Testing the explanatory variables for multi-collinearityusing a Variance Infla- tion Factor (VIF). (PDF) S3 Table. Testing the explanatory variables for heteroskedasticity. (PDF) S4 Table. Summary of the marginal effects of ordered probit model with sample selection in second stage decision making. (PDF) S5 Table. Ordered probit model of fisher data from Sablayan and Mamburao, Occidental Mindoro with inverse Mills ratio. (PDF) S6 Table. Ordered probit model of fisher data from Occidental Mindoro without inverse Mills ratio. (PDF) Acknowledgments The main author would like to thank the WWF-PPTST site coordinators in Occidental Min- doro and the Artesmar for their openness to provide information and kind assistance in con- ducting field surveys. The authors would also like to thank the reviewers for their critical comments and suggestions in improving this manuscript. This research was conducted through the BESTTuna programme at Wageningen University with funds from the Wagenin- gen University Interdisciplinary Research and Education Fund (INREF). S4 Table. Summary of the marginal effects of ordered probit model with sample selection in second stage decision making. (PDF) S5 Table. Ordered probit model of fisher data from Sablayan and Mamburao, Occidental Mindoro with inverse Mills ratio. (PDF) S1 File. Estimation of the sample size. (PDF) S1 Table. Data of individual fisher surveysin Occidental Mindoro fishery. (XLSX) S2 Table. Testing the explanatory variables for multi-collinearityusing a Variance Infla- tion Factor (VIF). (PDF) S2 Table. Testing the explanatory variables for multi-collinearityusing a Variance Infla- tion Factor (VIF). (PDF) Conclusion Our results indicate the overall importance of considering fisher capabilities in understanding decisions of fishers to participate in different kinds of FIPs. The individual firm capabilities stimulate fishers to participate in strongly market oriented top-down FIPs while collective capabilities enable fishers to improve their practices and to participate in bottom-up FIPs aimed at wider fishery and community development goals. These types of capabilities and FIP models have consequences for the inclusiveness of different fishers, for which three insights are generated in this study. First, the top-down FIPs appear to be more exclusive, given that there are a few numbers of fishers who have the capabilities and lower risk aversion to comply with its requirements. Second, bottom-up FIPs appear to be more inclusive, aiming to provide broad support to the development of fisher capabilities and the institutional support provided to them to improve their practices on the water. Third, the two FIP models exhibit competing inclusion when implemented in the same fishery, yet also exhibit potential complementarity in terms of delivering incentives and improving governance of the fishery. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 17 / 22 Writing – review& editing: FTZ PB SRB LD AOL. Writing – review& editing: FTZ PB SRB LD AOL. Author Contributions Conceptualization: FTZ PB SRB LD AOL. Conceptualization: FTZ PB SRB LD AOL. PLOS ONE \| DOI:10.1371/journal.pone.0163537 October 12, 2016 18 / 22 Fishers’ Decisions to Participate in FIPs Data curation: FTZ. 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https://openalex.org/W4229024551	https://www.intechopen.com/citation-pdf-url/79434	English	null	Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr Viral Infection	IntechOpen eBooks	2,022	cc-by	14,997	Abstract We demonstrated that in patients with an initially low level of induced IFN-γ, the production of this cytokine significantly increased in three months after the end of therapy. Keywords: Epstein-Barr virus, immunity, recombinant human interferon-γ, treatment, herpesvirus Keywords: Epstein-Barr virus, immunity, recombinant human interferon-γ, treatment, herpesvirus Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr Viral Infection Irina A. Rakityanskaya and Tatiana S. Ryabova Irina A. Rakityanskaya and Tatiana S. Ryabova Abstract Infection of Epstein-Barr virus (EBV) is about 90% among people over the age of 40. The EBV causes a chronic infection that is characterized by chronic or recurrent symptoms and persists for a long time. Recombinant interferon-gamma (IFN-γ) has high clinical and antiviral efficacy in the treatment of herpesvirus infections. 110 patients with chronic EBV infection were examined. The patients were divided into three groups for different treatment regimens: Group 1—IFN-γ therapy (15 injections of Ingaron i/m, 500,000 IU every other day); Infection of Epstein-Barr virus (EBV) is about 90% among people over the age of 40. The EBV causes a chronic infection that is characterized by chronic or recurrent symptoms and persists for a long time. Recombinant interferon-gamma (IFN-γ) has high clinical and antiviral efficacy in the treatment of herpesvirus infections. 110 patients with chronic EBV infection were examined. The patients were divided into three groups for different treatment regimens: Group 1—IFN-γ therapy (15 injections of Ingaron i/m, 500,000 IU every other day); Group 2—valaciclovir (Valtrex 500 mg 2 times/day, orally for 2 months); Group 3—valganciclovir (Valcyte 450 mg 2 times/day, orally for 2 months) and IFN-γ (10–20 injections of Ingaron i/m, 500,000 IU every other day). The best results were obtained in group 3–73.07% negative PCR. In this group, the combination of valganciclovir + IFN-γ was different. We showed that the efficacy of therapy in patients with chronic EBV is determined by the duration of INF-γ administration. We also determined spontaneous and induced production of IFN-α and -γ cytokines in serum and in lymphocyte culture. We demonstrated that in patients with an initially low level of induced IFN-γ, the production of this cytokine significantly increased in three months after the end of therapy. py ( j g , , y y); Group 2—valaciclovir (Valtrex 500 mg 2 times/day, orally for 2 months); Group 3—valganciclovir (Valcyte 450 mg 2 times/day, orally for 2 months) and IFN-γ (10–20 injections of Ingaron i/m, 500,000 IU every other day). The best results were obtained in group 3–73.07% negative PCR. In this group, the combination of valganciclovir + IFN-γ was different. We showed that the efficacy of therapy in patients with chronic EBV is determined by the duration of INF-γ administration. We also determined spontaneous and induced production of IFN-α and -γ cytokines in serum and in lymphocyte culture. 1. Introduction The development of immunodeficiency leads to the spread of persistent and/or chronic herpesvirus infection, in which the pathogen is not eliminated from the host’s body for months, years, or even throughout life. Each herpesvirus in the host organism has its own target tissue, where the virus persists with the ability to enter and exit the tissue using a developed strategic mechanism, which consists of the minimum expression of viral genes in a small number of infected cells or their elimination at the protein level. This allows the virus to evade the immune response and remain in very small quantities (1 infected cell per 5 ml of blood) with minimal impact on the patient’s body, remaining in it for the rest of its life. In this case, the immune response is unable to eliminate the infectious pathogen from the body. 1 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control 1.1 Epstein-Barr virus Epstein-Barr virus (EBV) or infectious mononucleosis virus, together with other herpesvirus infections, is a prototype of persistent viral infections characterized by latency. In the mid-1980s, the problem of chronic EBV infection or “chronic mono- nucleosis” attracted the attention of researchers. It has been shown that EBV causes the development of chronic or recurrent infectious mononucleosis-like symptoms in immunocompetent individuals, which persist for a long time and are character- ized by constant fatigue, headaches, myalgias, lymphadenopathy, subfebrile fever (37.1–37.3°С), hepatosplenomegaly. Additionally, gastrointestinal diseases may develop hematological, neurological, and skin lesions [1]. p g g EBV infection is accompanied by high production of IgG antibodies to viral capsid (VCA) and early antigens (EA) when compared with the control group, as well as very low production or absence of antibodies to Epstein-Barr virus nuclear antigen (EBNA) [2]. That is why it was suggested that the cause of the syndrome is a chronic EBV infection [3]. However, some patients may not have abnormally high antibody titers associated with EBV [4]. According to data published by Kanegane et al., the severity of the disease directly correlates with the level of EBV DNA copies number in serum or plasma [5]. In later studies, it was shown that an increased level of EBV DNA in the blood is a more specific criterion for chronic EBV than the levels of antibodies to EBV [6]. In 1983, Hellman et al. first proposed an abbreviation for this syndrome “Chronic Active EBV infection” (CAEBV) [7]. As a result of the analysis of the literature, including work on chronic EBV as well as work on herpesvirus infections, immunodeficiencies, and three types were identified based on clinical, pathological, and virological data characteristic of this syndrome [8, 9]: 1.Chronic fatigue syndrome of unknown etiology (CFS), which is characterized by profound, debilitating fatigue and a combination of symptoms leading to a significant decline in occupational, personal, social, and educational status; 3.Severe CAEBV (SCAEBV). EBV is known to spread through contact with saliva and penetrate the epithe- lium that lines the nasopharynx. Waldeyer’s ring, which includes the adenoids, tonsils, and the lymphoid system surrounding the nasopharyngeal region, forms a continuous structure called lymphoepithelium [10]. The virus infects epithelial cells, replicates, then is released, followed by infection of resting naive B cells in nearby areas by activating latent proteins (six EBV nuclear antigens (EBNAs: EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP), two latent membrane proteins (LMPs: LMP1, LMP2) and viral anti-apoptotic protein BHRF1) encoded by the growth program. As a result, the cell becomes a proliferating lymphoblast (lymphoblastic burst). This program leads to the expansion of EBV-infected B cells in the lymphoid tissue of the oropharynx and the appearance of infected B cells in the blood. In some infected B cells, EBV inhibits its growth transformation pro- gram, allowing the cells to enter the memory B cell pool, while the virus persists as a truly latent infection devoid of viral gene expression. Therefore, the target of the incoming virus is a resting naive B cell, which becomes infected as it passes through the epithelium. Naive B cells are continuously recirculated throughout the body, extravasating from the peripheral circulation to secondary lymphoid tissue through specialized structures called high endothelial vesicles (HEVs) located in the lymphoepithelium. Naive B cells migrate to the mantle zone of the follicles below 2 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 the epithelium and remain there for several days, and then return to the circulation. Infection of new naive B cells occurs in the intraepithelial layer, and not in the mantle zone [11], that is, when an infected B cell enters the follicle, it is already a blast and cannot migrate to the mantle zone. EBV infects cells through the interac- tion of viral glycoproteins gp350/220 with CD21 and gp42/gH/gL with class II HLA in a B cell. Thus, memory B cells are a place of long-term viral persistence, where the virus can remain throughout the patient’s life, because immunological memory is formed, and the virus ceases to be pathogenic for the host since the genes that induce cell proliferation and contribute to the development of neoplastic disease are disabled. 3.Severe CAEBV (SCAEBV). However, EBV can infect T and NK (natural killer) cells of the tonsils [12] and peripheral blood [13]. Expression of CD21 on T cells [14] and NK cells can be induced by trogocytosis and the formation of an immunological synapse that occurs when EBV contacts B cells, which leads to a possible EBV infection [15]. p It has been shown that the level of infected B cells in the population ranges from 5 to 3000 in every 107 memory B cells, both in peripheral blood (on average 110/ 107) and in Waldeyer’s ring (average value 175/107), then there is a virus evenly distributed throughout the ring. Thus, the level of infected cells is similar between peripheral blood and Waldeyer’s ring, but 20 times lower than in other lymphoid tissue (spleen and mesenteric lymph node) [16]. The total body load in humans amounts to 104–107 (on average 0.5/106) infected memory B-cells, representing a small, stable, and most critically “safe” pool of infected cells, which guarantees long-term persistence. Only about 1% of these cells are found in the peripheral blood. One latently infected memory B cell in the amygdala can differentiate into a plasma cell and secrete a virus that infects epithelial cells. The virus constantly seeps into the oral cavity, where it mixes with saliva for about 2 minutes before swallowing. Thus, the oral cavity is a reservoir for EBV flow and not a static reservoir. About 250 cells begin to replicate in the Waldeyer ring at any given time. The oral cavity and peripheral blood are important anatomical sites for the locali- zation and persistence of EBV infection. These two compartments are connected by oropharyngeal lymphoid tissue such as the lingual, palatine, and pharyngeal tonsils. EBV-infected B cells can re-enter the tonsils, where memory lymphocytes express characteristic sets of adhesion receptors, through which they are able to return to target organs, where they first encountered antigens. Thus, EBV-infected cells can release viral particles through lytic replication, reinfect cells in lymphoepithelial tissue, and subsequently release viruses into the oral cavity [17]. The release of viruses into plasma from different anatomical sites indicates that different viral strains can persist in different tissue compartments. Therefore, EBV can be detected in the tissues of various anatomical structures of the human body. 3.Severe CAEBV (SCAEBV). Despite the fact that EBV is an oncogenic virus the vast majority of EBV- Despite the fact that EBV is an oncogenic virus, the vast majority of EBV- infected people do not suffer from any long-term consequences. This is due to the antiviral immune response that develops during primary infection with EBV, and further supports subsequent lifelong control to ensure the mutual coexistence of the virus and its host. Early control of EBV infection is associated with the expansion of innate immune cells (primarily NK cells) and CD8+ and CD4+ T cells, specific for a wide range of EBV proteins expressed during the lytic and latent stages of viral infection. Patients with persistent EBV infection develop a specific CD8+ T-cell response to antigens of the lytic and latent cycles, the former being more frequent. An individual lytic epitope-specific response can account for up to 2% of the total population of CD8+ T cells. The response to immediate-early antigens dominates the response to early antigens, and the response to late expressed antigens rarely develops [18]. CD8+ T cells play a major role in the formation of responses to proteins EBNA3A, 3B, and 3C. A less specific immune response develops against EBNA1, EBNA2, and LMP2. Individuals expressing HLA-B3801 have been shown 3 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control to have strong responses to the EBNA2 epitope, and carriers of HLA-A0203 have strong responses to the epitope from EBNA-LP. In persistently infected individuals the EBV-specific T cell pool contains resting antigen-expressed T cells that are not active and do not proliferate. Lymphoid markers CCR7 and CD62L, specific for the latent antigen, are expressed on T cells. The phenotype, functional profile, and clonotypic composition of TCRs specific for CD8+ T cells remain stable for many years [19]. The EBV-specific CD4+ T cell response in healthy carriers is much weaker and may be 10 times less pronounced than the CD8+ T cell response to the same antigen. EBV-specific CD4+ memory T cells share the same phenotype regardless of whether they are specific for latent or lytic antigens. CD4+ T cells do not express perforin and granzyme, and upon ex vivo stimulation, the cytokine polyfunctionality of cells increases, and TNF-alpha production predominates [20]. NK-T cells are a conserved population of congenital T cells expressing the semi- invariant Va24-Ja18/Vβ11 T cell receptor. 3.Severe CAEBV (SCAEBV). Only one study evaluated the frequency of NK T cells in the blood during EBV infection, and it was shown that the number of NK T cells was increased in the first month of infection. A change in cellular phenotype and function was noted with an increase in the content of CD56 (bright) with a high ability to destroy EBV-infected cells. NK-T cells play an important role in the control of primary EBV infection by eliminating infected B cells and increas- ing the antigen-specific response of T cells through the release of immunomodula- tory cytokines [21]. It has been shown that patients with primary y y p p y immunodeficiency are predisposed to the development of EBV-associated disease. The presence of NK T cells reduces the EBV transformation of B cells in vitro. With EBV infection of blood lymphocytes, the previous depletion of NK T cells leads to both an increase in the number of B cells infected with EBV and an increase in the total viral load in culture. It has been suggested that NK T cells play a role in the early immune recognition of newly EBV-infected B cells [22]. 1.2 Interferon-γ IFN-γ achieves this by stimulating characteristic Th1-effector mechanisms: innate cell-mediated immunity (through the activation of NK cell effector functions), specific cytotoxic immunity (through the interaction of T cells with APC), and macrophage activation. IFN-γ increases the content of lymphocytes and leads to their long-term persistence in the tissue, induces activation of the complement cascade and acute phase response, plays a role in the switch of IgG class production, and has a direct antiviral effect [24]. Nor- mally, in the early stages of the host’s immune response, IFN-γ production by NK cells, CD4+ T (Th1) cells, and CD8+ T cells is aimed at improving antigen recogni- tion in APCs such as macrophages and dendritic cells. IFN-γ is one of the key cytokines that differentiate naive CD4 cells into Th1 effector T cells, which produce the main mediators of cellular immunity against viral and intracellular bacterial infections [25]. Together, IFN-γ and IL-12 generate a very strong Th1 response. Th1 cell-mediated immunity and Th2 cell-mediated humoral immunity are modulated by IFN-γ, which affects the differentiation of naive T cells into Th1 or Th2 cells. y γ, When activated, almost all CD8+ T cells, NK cells, and Th1 lymphocytes pro- duce IFN-γ, which stimulates cytokine activity and increases the expansion of low avid NK cells. Of all the interferons/cytokines of the Th1 response, IFN-γ is most strongly correlated with the Th1 response [26]. The effects induced by IFN-γ lead to increased immune surveillance. In addition, IFN-γ blocks the production of IL-4, an inducer of Th2 cell differentiation and proliferation. The synergistic effects of IL-21, IL-18, and IL-15 increase IFN-γ production. The most potent regulator of IFN-γ production is IL-15 compared to IL-21 in human NK and T cells. The cytokines IL-15 and IL-18 are produced by macrophages, while IL-21 is mainly produced by acti- vated T cells. IFN-γ increases the expression of the HLA (major histocompatibility complex) class I and II antigen by increasing the expression of subunits, increasing the expression and activity of proteasomes, resulting in increased sensitivity of the host to an infectious pathogen and an increased ability to identify and respond to this pathogen [26]. Thus, IFN-γ has many important immunostimulatory and immunomodulatory effects. y With the development of inflammation, a high level of IFN-γ leads to the activation of both canonical and non-canonical pathways. 1.2 Interferon-γ Interferons (IFNs) are important biological regulatory proteins called cytokines and mediators of cellular homeostatic reactions that are produced in response to viral infection inhibiting the replication of a wide range of DNA and RNA viruses, thereby creating negative feedback. Inhibition of the viral replication cycle is carried out with the help of the synthesis of viral polypeptides [23]. When IFNs are administered in vivo, the level of viremia decreases, that is, interferons can be used as antiviral drugs, and the antiviral effect is mediated both by the immune system itself and by intracellular antiviral mechanisms. All types of IFNs inhibit more than one step in the viral life cycle: viral entry and decay, viral mRNA transcription, viral protein synthe- sis, viral genome replication, and progeny assembly and isolation of virions. According to the amino acid sequence IFNs are divided into three types: I II and III According to the amino acid sequence, IFNs are divided into three types: I, II, and III. IFN-γ is the only representative of type II IFN. It is structurally unrelated to type I IFNs, binds to another receptor and is encoded by a separate chromosomal locus. Type II IFN or immune IFN—IFN-γ is a highly pleiotropic cytokine, secreted not in response to viral infection, but indirectly by mitogen-activated T cells and NK cells, which are the primary producers of IFN-γ during the innate and adaptive phases of the immune response to viral infection. Other cells such as B cells, NK T cells, and professional antigen-presenting cells (APCs) have now been shown to secrete IFN- γ. The production of IFN-γ by monocytes/macrophages and dendritic cells acting locally is important in cell activation [24]. y IFN-γ plays an important role in the activation of macrophages for the produc- tion of tumor necrosis factor-α (TNF-α), increases macrophage phagocytosis and microbicidal activity, the formation of active nitrogen and oxygen intermediates, 4 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 including superoxide radicals - nitric oxide and hydrogen peroxide, which are powerful cytotoxic effectors, stimulates the Th1-T cell response and has a strong inflammatory activity. IFN-γ is the main product of Th1 cells and further shifts the immune response towards the Th1 phenotype. 1.2 Interferon-γ In the canonical signaling pathway, IFN-γ dimerizes and binds to two IFN-γ receptors, which are composed of two different ligand-binding chains: high-affinity IFNGR-1 (α) with high expres- sion and two signal-transforming low-affinity IFNGR-2 (β),—with related signaling mechanisms. The IFNGR1 and IFNGR2 chains belong to the class II cytokine recep- tor family. The IFNGR2 chain limits sensitivity to IFN-γ and the IFNGR1 chain is usually in excess. But the expression level of IFNGR2 can be tightly regulated depending on the state of cell differentiation or activation. Receptors are expressed on the surface of almost all cell types. The expression level is determined by the cell type and its activation status. Initially, IFN-γ binds to IFNGR1, and the formed IFN- γIFNGR1 complex facilitates its binding to IFNGR2, then downstream signaling pathway events are initiated [27]. IFN-γ gene transcription is induced through several mechanisms. The most studied response to IFN-γ, mediated by STAT-1- containing transcription factor GAF (gamma-activated factor), which is activated by tyrosine kinases Jak1 and Jak2 and binds to the gamma-activating sequence GAS (Gamma Activating Sequence), which is present in the promoter regions of many genes. As a result of gene activation, the formation of cellular immune response to a viral infection begins [28]. The JAK/STAT pathway is the main signaling pathway initiated by IFN-γ stimulation. Further, IFN-γ, together with one of its receptor 5 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control subunits IFNGR1 and pSTAT1, is translocated into the cytoplasmic domain in com- bination with endocytosis and induces gene expression by binding to GAS elements in the promoter region of inducible IFN genes [29]. When viruses inhibit the func- tions of STAT1 molecules, IFN-γ can independently induce a noncanonical signaling pathway [30]. That is, IFN-γ is capable of inducing gene expression in STAT1/ bone marrow macrophages, suggesting that IFN-γ acts independently of STAT-1 or in an alternative non-canonical manner. Typically, activation of noncanonical pathways occurs later, after STAT1 activation. However, there is evidence that noncanonical pathways can be activated in the absence or presence of STAT1 in a dependent manner [31]. The IFN-γ and IFN-α/β signaling pathways intersect at several levels, partially overlap, which allows cross-interaction of certain functions within the cell. This crossover mechanism is relevant because in vivo cells are not stimulated in isolation by a single cytokine, but rather a cytokine cocktail that induces gene expression through the integration of multiple signaling pathways. 1.3 The use of interferon-γ in the treatment of herpesvirus infections In recent years, numerous works have been published in the world on the treatment of herpesvirus infections with recombinant IFN-γ, showing high clinical and antiviral efficacy [27, 37–39]. IFN-γ demonstrated a 7–10 times more potent antiviral effect than IFN-α or -β. When IFN-γ is added at the 3–4th days after infecting, there is a decrease in EBV-induced B cell proliferation and immunoglob- ulin secretion, while the addition of IFN-α and -β has an effect only within 24 h. Lotz et al. Found that EBV-infected cells can be regulated by all IFNs at an early stage. Subsequently, there comes an intermediate period when only IFN-γ is able to directly influence EBV-induced B-cell responses. In the third phase, B-lymphocytes become insensitive to the direct action of all IFNs and are exposed only to cytotoxic cells [40]. In 2002 the introduction of recombinant IFN-γ, as well as IFN-β, showed high efficiency of inhibition of replication of the herpes simplex virus type 1 (HSV- 1) [41]. That is, the high level of inhibition achieved by the administration of exogenous IFN-γ was the result of a synergistic interaction with endogenous IFN-α/β, which is produced locally in response to HSV-1 infection. Other researchers revealed that IFN-β and IFN-γ interact synergistically, blocking viral DNA synthesis and nucleocapsid formation in HSV-1 infected cells, without affect- ing the viability of the host cells. Thus, the authors concluded that IFN-mediated suppression of HSV-1 replication plays the role of the main mechanism by which the host immune system limits the spread of infection in vivo [42]. In a double- blind, placebo-controlled study, it was shown that the introduction of recombinant IFN-γ three times a week subcutaneously reduces the incidence of severe infections in patients with various genetic types of chronic granulomatous disease [43]. p g yp g In the Russian Federation, the only IFN-γ preparation has been registered under the trade name Ingaron, developed by SPP PHARMACLON Ltd. via the microbio- logical synthesis in a recombinant E. coli strain and purified by column chromatog- raphy. The drug consists of 144 amino acid residues, devoid of the first three of them (Cys-Tyr-Cys), replaced by Met. 1.2 Interferon-γ When infected with a virus, IFN-γ can induce apoptosis by regulating Fas ligands to remove virus-infected cells, enhancing the expression of type I IFNs, pro- inflammatory cytokines, and chemokines by endothelial, epithelial cells and fibro- blasts to attract macrophages, neutrophils, and T cells to the sites of infection [32]. IFN-γ can also initiate the expression of dsRNA-specific adenosine deaminase (ADAR), which inhibits viral replication by editing or disrupting the translation of viral proteins [33]. p Virus infection of a cell begins with the attachment of the virus to the surface of the host cell through a receptor and/or through cell membrane molecules such as glycans. Viruses can release their genomes directly into the cell after fusion of its membrane with the plasma membrane, while other viruses enter cells through cellular endocytosis, which allows the virus to release the core virion containing the viral genome directly into the cytoplasm [34]. The isolated genome, either naked or associated with viral proteins, moves to certain regions of the cytoplasm or nucleus for its replication [35]. IFN-γ can inhibit the entry of the virus from the endosome into the cytoplasm. y p Virus replication is the primary goal of the virus life cycle [36]. Suppression of any stage of the life cycle can lead to suppression of viral genome replication during viral infection. IFN-γ is a potent antiviral cytokine that interferes with various stages of the viral life cycle in stimulated cells using the following mechanisms [35]: 1.Inhibition of the viral penetration, both at the extracellular and intracellular stages, by controlling the expression and/or distribution of the receptors necessary for the penetration of the virus; 2.Inhibition of the viral replication by disrupting the replication niche of the virus; 3.Disruption of gene expression, interfering with translation; 4.Violation of stability by interfering with nucleocapsid assembly; 5.Violation of virus shedding by breaking the disulfide bond of the required participant in cellular interaction; 7.IFN-γ can also inhibit the penetration of the virus at the stage of transfer of the invading virus from the endosome into the cytoplasm [35]. 6 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 1.3 The use of interferon-γ in the treatment of herpesvirus infections The purpose of this study is to evaluate the efficacy of IFN-γ therapy for the content of the number of EBV DNA copies in saliva samples by the Real-time PCR method, for the dynamics of INF-α and INF-γ production (spontaneous, serum, and induced levels) and the clinical picture in patients suffering from chronic Epstein- Barr virus infection (CEBVI) one and three months after the end of therapy. 2. Material and methods 2.1 Schemes of therapy Table 1. Ch The study procedures were in accordance with the Good Clinical Practice (GCP) guidelines and ethical principles of the Declaration of Helsinki. The study was approved by the Ethics Committee of Fresenius Medical Care (Dialysis Center St. Petersburg, Russia). Written informed consent was obtained from all participants before the study was initiated. 2.1 Schemes of therapy All patients were divided into three groups for different therapy regimens • The first group consisted of 51 patients (from 22 to 49 years old) who received IFN-γ therapy (500,000 IU every other day, intramuscular injections (i/m)). The total course was 15 injections; • The second group consisted of 42 patients (from 22 to 48 years old) who received prolonged therapy with a drug from the group of acyclic natural nucleosides - valaciclovir (Valtrex 500 mg 2 times/day, orally) for two months; • The third group consisted of 46 patients (from 19 to 52 years old) who received prolonged therapy with a synthetic nucleoside analog of guanosine—valganciclovir 7 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control (Valcyte 450 mg 2 times a day, orally) for 2 months in combination with IFN-γ (10–20 intramuscular injections of Ingaron 500,000 IU every other day). Previously, all patients in this group, as prescribed by a doctor or independently (often repeatedly), received therapy with drugs from the group of acyclic natural nucleosides, including valaciclovir for short courses (7–10 days). There was no pronounced clinical and laboratory positive effect from the previous therapy, for this reason, these patients have prescribed valganciclovir in combination with IFN-γ. To assess the efficacy, a comparative analysis of the amount of EBV DNA in saliva samples was carried out one month after the end of the treatment course. Clinical complaints were compared for the patients of Group 1 in one and three months after the treatment course. Patient groups and therapy are presented in Table 1. Patient group Therapy in the main groups Subgroup of patients Therapy in subgroups 1st group (n = 51) Ingaron 500,000 IU every other day, i/m. Course of 15 injections — — 2nd group (n = 42) Valtrex 500 mg 2 times/ day, 2 months — — 3d group (n = 46) Valcyte 450 mg 2 times/ day, 2 months + Ingaron 3А group (n = 22) Valcyte 450 mg 2 times/day (2 months) + Ingaron 500,000 IU every other day, i/m. Course of 10 injections 3В group (n = 24) Valcyte 450 mg 2 times/day (2 months) + Ingaron 500,000 IU every other day, i/m. Course of 20 injections Table 1. Characteristics of therapy in patient groups. Table 1. Characteristics of therapy in patient groups. 2.3 Statistical analysis Statistical analysis of the obtained results was carried out using the statistical software package IBM SPSS Statistics, version 26 (Armonk, NY: IBM Corp.). Group results are presented as mean standard error of the mean (SEM). Statistical processing of the results was carried out using parametric (Pearson’s method) and nonparametric (Kendall’s tau (τ)) criteria. To check the condition of independence of observations, linear regression analysis (with the calculation of the coefficient of determination (R Square) and the Durban-Watson criterion) and analysis of vari- ance (ANOVA Analisis of Variance) with the calculation of the Fisher criterion (F) were carried out to test the significance of the model. The standardized coefficient ß was calculated with 95% Confidence Interval (95% CI). The critical level of signif- icance of the difference in indicators was taken equal to 0.05. 2.2 Survey methods Clinical research methods included the collection of anamnesis, data on previous immuno- or antiviral therapy, and the presence of concomitant diseases. The clini- cal condition of patients was assessed according to the generally accepted method, including objective data and registration of patient complaints at the time of exam- ination. The severity of patient complaints was recorded using a subjective assess- ment scale on a 3-point scale (0—no symptoms, 1—mild symptoms, 2—moderate severity of symptoms, 3—significant severity of symptoms). y y p g y y p Diagnosis of CEBVI was based on clinical data and positive results of EBV DNA studies in saliva samples conducted by polymerase chain reaction (PCR) with real- time hybridization-fluorescence detection. The test systems “AmpliSens EBV/ CMV/HHV6-screen-FL” (FBSI “Central Research Institute of Epidemiology”, Rus- sia) were used. The unit of measurement used to estimate the viral load during DNA extraction from saliva is the number of copies of EBV DNA per ml of sample. According to the instructions this indicator is calculated using the formula: Number p p p According to the instructions, this indicator is calculated using the formula: Number 8 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 of DNA copies = CDNA 100, where CDNA is the number of copies of the viral DNA in the sample. The analytical sensitivity of the test system is 400 copies/ml. DNA in the sample. The analytical sensitivity of the test system is 400 copies/ml. We studied the dynamics of IFN-α and -γ production before the initiation of IFN-γ therapy and 1 and 3 months after the end of the course. Determined the level of IFN-α and IFN-γ in the blood serum, as well as the spontaneous and induced production of these cytokines in the culture of blood lymphocytes. Newcastle dis- ease virus (NDV) (obtained at the L.A. Tarasevich State Institute of Culture, St. Petersburg) with an infectious titer of 8 lg EID/0.2 ml in a volume of 8 μl/hole and Phytohemagglutinin (PHA-P) (PanEko, Russia) at the dose of 10 μg/ml were used as inducers of IFN-α and -γ production respectively. The quantitative content of cytokines was determined in the serum and supernatant of a 24-h whole blood culture by solid-phase ELISA using the test systems “alpha-Interferon-IFA-BEST” and “gamma-Interferon-IFA-BEST” (JSC “VectorBest”, Russia). Reference values for spontaneous, serum, and induced production of IFN-α and IFN-γ are provided by the manufacturer of the test systems. 3. Results The examination was carried out in 139 patients with СEBVI: 86 women and 53 men with average age of 35.27 1.28 years. The duration of CEBVI from the appearance of the first complaints to laboratory confirmation and the diagnosis was 2.23 0.21 years. 98 (7.720%) and 27 (24.54%) patients suffered in childhood from chronic tonsillitis with no response to antibiotic therapy and infectious mononucle- osis respectively. All patients underwent differential diagnosis of CEBVI with other viral infections (HIV, viral hepatitis, cytomegalovirus infection, toxoplasmosis), helminthic invasions, autoimmune diseases associated with EBV infection. CEBVI is characterized by a long course and frequent relapses with clinical and laboratory signs of viral activity, described in detail in the literature [44–46]. Patients worried about prolonged subfebrile condition (37.1–37.3°C), weakness, unmotivated fatigue, excessive sweating (especially at night), a constant feeling of discomfort and/or pain in the throat, lymphadenitis, swelling of the nasal mucosa with abundant drainage mucus, stomatitis. Some patients have a cough, skin rashes, arthralgia, pain in the muscles of the trunk and extremities are possible. There may be manifestations of conjunctivitis, otitis media. Neurological disorders develop headaches, memory and sleep disorders, decreased concentration, irritability, tear- fulness, a tendency to depression. Perhaps an increase in internal organs (according to ultrasound, hepato- or splenomegaly) and feeling of heaviness in the right hypochondrium. Also, patients complain of frequent colds, the addition of other herpesviral infections. In the history of such patients, long-term stressful situations, 9 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control psychoemotional and physical overloads often take place, against the background of which the patient’s condition worsens. 3.1 Comparative analysis of the efficacy of antiviral therapy In all patients (N = 139) CEBVI was confirmed by PCR reaction in saliva samples. Table 2 shows the results of a comparative analysis of the dynamics of the number of EBV DNA copies in groups of patients who received different therapy regimens. Patient group Number of copies/ml before therapy Number of copies/ml after therapy 1st group IFN-γ (N = 51) 294630.59 72210.69 154786.97 18671.15 (N = 36) in 15 patients—0.00 copies (29.41%) 2nd group valaciclovir (2 months) (N = 42) 278857.24 44608.15 47108.18 25928.62 (N = 30) in 12 patients—0.00 copies (28.57%) 3d group valganciclovir (2 months) + IFN-γ (N = 46) 425250.00 62697.09 35934.50 33764.56 (N = 13) in 33 patients—0.00 copies (71.74%) Table 2. Dynamics of the number of EBV DNA copies one month after the end of antiviral therapy in patients with CEBVI. Patient group Number of copies/ml before therapy Number of copies/ml after therapy 1st group IFN-γ (N = 51) 294630.59 72210.69 154786.97 18671.15 (N = 36) in 15 patients—0.00 copies (29.41%) 2nd group valaciclovir (2 months) (N = 42) 278857.24 44608.15 47108.18 25928.62 (N = 30) in 12 patients—0.00 copies (28.57%) 3d group valganciclovir (2 months) + IFN-γ (N = 46) 425250.00 62697.09 35934.50 33764.56 (N = 13) in 33 patients—0.00 copies (71.74%) Table 2. Dynamics of the number of EBV DNA copies one month after the end of antiviral therapy in patients with CEBVI. From the presented data in the 1st group after Ingaron therapy, only 15 (29.41%) patients had negative PCR results in saliva samples. In the 2nd group of patients taking valaciclovir negative PCR results were obtained in 12 (28.57%) patients. In group 3, one month after taking the combination therapy valganciclovir + Ingaron, 33 (71.74%) patients obtained negative PCR results in saliva samples. p g p The patients of the 3rd group, according to the combination therapy were distributed as follows: • 3A subgroup—22 patients received Valcyte 900 mg/day (2 months) + Ingaron 10 injections of 500,000 IU every other day; • 3A subgroup—22 patients received Valcyte 900 mg/day (2 months) + Ingaron 10 injections of 500,000 IU every other day; • 3B subgroup—24 patients received Valcyte 900 mg/day (2 months) + Ingaron 20 injections of 500,000 IU every other day. Table 3 shows the results obtained in these subgroups. 3.1 Comparative analysis of the efficacy of antiviral therapy Therapy scheme (Group 3) Number of DNA copies/ ml before therapy Number of DNA copies/ml after therapy Subgroup 3A: Valcyte 900 mg/day (2 months) + Ingaron 500,000 IU 10 (N = 22) 334086.00 95214.02 In 12 patients—0.00 copies; (54.54%) in 12 patients—6285.57 2823.61 Subgroup 3B: Valcyte 900 mg/day (2 months) + Ingaron 500,000 IU 20 (N = 24) 381745.32 161946.09 In 21 patients—0.00 copies; (87.50%) in 3 patients—123469.51 46615.32 Table 3. Dynamics of the number of EBV DNA copies one month after the end of combined antiviral therapy in the 3rd group of patients. • 3B subgroup—24 patients received Valcyte 900 mg/day (2 months) + Ingaron 20 injections of 500,000 IU every other day. Table 3 shows the results obtained in these subgroups. Table 3 shows the results obtained in these subgroups. Therapy scheme (Group 3) Number of DNA copies/ ml before therapy Number of DNA copies/ml after therapy Subgroup 3A: Valcyte 900 mg/day (2 months) + Ingaron 500,000 IU 10 (N = 22) 334086.00 95214.02 In 12 patients—0.00 copies; (54.54%) in 12 patients—6285.57 2823.61 Subgroup 3B: Valcyte 900 mg/day (2 months) + Ingaron 500,000 IU 20 (N = 24) 381745.32 161946.09 In 21 patients—0.00 copies; (87.50%) in 3 patients—123469.51 46615.32 Table 3. Dynamics of the number of EBV DNA copies one month after the end of combined antiviral therapy in the 3rd f i 10 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 From the data presented in Table 2 one can see that in the total 3rd group of patients, there is a reliably positive dynamics of the number of DNA copies the decrease in a month after combination therapy. In subgroup 3A, negative PCR results were obtained in 54.54% of patients. The best result was observed in the patients of subgroup 3B (in 87.50% of patients) who received 20 injections of IFN-γ 500,000 IU every other day, in combination with valganciclovir. That is, the posi- tive result of this therapy regimen is due not so much to the combination of medicines, but to the amount and duration of IFN-γ administration. 3.2 Dynamics of INF-α and INF-γ production After the comparative analysis of the efficacy of different regimens of CEBVI therapy, we analyzed the dynamics of INF-α and INF-γ production (spontaneous, serum, and induced) in the culture of lymphocytes in the first group of patients (N = 51) before the start of therapy with IFN-γ, after one and three months after the end of therapy. We also assessed the dynamics of clinical complaints in these patients after IFN-γ therapy. Tables 4 and 5 present the data obtained. Research indicator IFN-α level (pg/ ml)before therapy IFN-α level (pg/ml) after 1 month of therapy IFN-α level (pg/ml) after 3 months of therapy p 1 2 3 Spontaneous IFN-α 3.76 0.58 5.80 4.02 3.85 19.24 Р1,2 = 0.345 Р2,3 = 0.435 Р1,3 = 0.359 Serum IFN-α 5.09 1.47 4.21 0.70 5.57 1.20 Р1,2 = 0.289 Р2,3 = 0.202 Р1,3 = 0.380 Induced IFN-α 296.78 127.43 578.154 129.46 294.78 60.67 Р1,2 = 0.284 Р2,3 = 0.360 Р1,3 = 0.145 Table 4. Dynamics of IFN-α production before the start, one and three months after therapy in the 1st group of CEBVI patients (N = 51). Research indicator IFN-γ level (pg/ml) before therapy IFN-γ level (pg/ml) after 1 month of therapy IFN-γ level (pg/ml) after 3 months of therapy p 1 2 3 Spontaneous IFN-γ 2.07 0.26 2.57 0.75 2.00 0.57 Р1,2 = 0.34 Р1,3 = 0.36 Р2,3 = 0.57 Serum IFN-γ 1.85 0.14 5.57 1.20 2.10 0.68 Р1,2 = 0.024 Р1,3 = 0.21 Р2,3 = 0.38 Induced IFN-γ 1862.72 624.52 2487.96 437.73 4308.12 3053.77 Р1,2 = 0.38 Р1,3 = 0.38 Р2,3 = 0.27 Table 5. Dynamics of IFN-γ production before the start, one and three months after therapy in the 1st group of CEBVI patients (N = 51). Table 4. Table 4. Dynamics of IFN-α production before the start, one and three months after therapy in the 1st group of CEBVI patients (N = 51). Research indicator IFN-γ level (pg/ml) before therapy IFN-γ level (pg/ml) after 1 month of therapy IFN-γ level (pg/ml) after 3 months of therapy p 1 2 3 Spontaneous IFN-γ 2.07 0.26 2.57 0.75 2.00 0.57 Р1,2 = 0.34 Р1,3 = 0.36 Р2,3 = 0.57 Serum IFN-γ 1.85 0.14 5.57 1.20 2.10 0.68 Р1,2 = 0.024 Р1,3 = 0.21 Р2,3 = 0.38 Induced IFN-γ 1862.72 624.52 2487.96 437.73 4308.12 3053.77 Р1,2 = 0.38 Р1,3 = 0.38 Р2,3 = 0.27 Table 5. Dynamics of IFN-γ production before the start, one and three months after therapy in the 1st group of CEBVI patients (N = 51). le 4. amics of IFN-α production before the start, one and three months after therapy in the 1st group of CEBVI ents (N = 51) Table 4. Dynamics of IFN-α production before the start, one and three months after therapy in the 1st group of CEBVI patients (N = 51). Table 5. Dynamics of IFN-γ production before the start, one and three months after therapy in the 1st group of CEBVI patients (N = 51). 11 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control One month after the end of therapy with IFN-γ, a tendency to an increase in the spontaneous production of IFN-α was revealed (statistically insignificant), but after three months the values returned to the initial values. Serum IFN-α production did not change after one and three months, remaining within the normal range. There was a tendency to an increase in the induced production of IFN-α one month after the end of therapy, followed by a normalization of the level after three months. Thus, IFN-γ had no significant effect on IFN-α production in the general group of patients after one and three months of therapy. y From the data presented in Table 4, it follows that in the group of patients a month after the end of therapy with IFN-γ, the serum (p = 0.024) production of IFN-γ increased, and after three months the serum level practically returned to the initial value (p = 0.57). The level of spontaneous production one and three months after the end of therapy did not change significantly. Table 4. Induced production of IFN-γ also tended to increase one and three months after the end of therapy without significant dynamics. g y When analyzing the initial data of the level of induced IFN-γ, it was found that these values sharply differed in patients, i.e. from the lower to the upper limit values of the reference (281-4335 pg/ml). pg In this regard, the group of patients (N = 51) was divided into 2 groups in accordance with the induced production of IFN-γ before the start of therapy: • 1st subgroup (N = 30)—the level of induced IFN-γ closer to the upper limit of the reference values (2706.00 1058.94 pg/ml); • 2nd subgroup (N = 21)—the level of induced IFN-γ closer to the lower limit of the reference values (287.20 64.65 pg/ml). • 2nd subgroup (N = 21)—the level of induced IFN-γ closer to the lower limit of the reference values (287.20 64.65 pg/ml). Figure 1 shows the data on the dynamics of the induced IFN-γ production in these groups of patients. The results of the study showed that after the course of therapy with IFN-γ in the 1st subgroup, the content of induced IFN-γ had a tendency to a gradual decrease, while in the 2nd subgroup there was a significant increase in the level of induced IFN-γ three months after therapy (p = 0.027). At the same time, the values of IFN-γ levels in both groups remained within the reference values. Figure 2 shows the results of the dynamics of the spontaneous level of IFN-γ before and after IFN-γ therapy in both subgroups. Figure 1. Dynamics of the level of induced IFN-γ before the start, one and three months after IFN-γ therapy in patients with CEBVI in the subgroups 1 and 2. Figure 1. Dynamics of the level of induced IFN-γ before the start, one and three months after IFN-γ therapy in patients with CEBVI in the subgroups 1 and 2. 12 Figure 2. Dynamics of the level of spontaneous IFN-γ before the start, one and three months after IFN-γ therapy in patients with CEBVI in subgroups 1 and 2. Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Figure 2. Table 4. Dynamics of the level of spontaneous IFN-γ before the start, one and three months after IFN-γ therapy in patients with CEBVI in subgroups 1 and 2. Figure 3. Dynamics of the level of serum IFN-γ before the start, one and three months after IFN-γ therapy in patients with CEBVI in subgroups 1 and 2. Figure 3. Dynamics of the level of serum IFN-γ before the start, one and three months after IFN-γ therapy in patients with CEBVI in subgroups 1 and 2. The presented data show that the values of the spontaneous level of IFN-γ in both groups significantly increased after one month. After three months, there was a trend towards a decrease in the spontaneous level of IFN-γ in both groups, while remaining above the baseline values. However, these values in both groups did not differ from the reference values (0–6 pg/ml). Figure 3 shows the dynamics of serum IFN-γ levels after therapy in both groups of patients. py g p p The results of the research showed that in both subgroups the increase in serum IFN-γ production was significant one month after the end of therapy (p = 0.03 and p = 0.02, respectively). In three months after the treatment course, there was a tendency to a slight decrease in serum IFN-γ, while the data obtained did not differ from the baseline (before the start of therapy levels) and from the reference values provided by the manufacturer of the test system (0–10 pg/ml). 3.3 Dynamics of clinical complaints The next stage of the work was the analysis of the frequency of the main clinical complaints in patients of both subgroups before the start, one and three months after IFN-γ therapy (Table 6). From the data in Table 6, one can see that in one and three months after the therapy with IFN-γ in patients of the 1st group there was a significant decrease in 13 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control Frequency of complaints (%) Before therapy (n = 30) after 1 month of therapy after 3 months of therapy p 1 2 3 Subfebrile temperature 83.33 30.76 30.76 Р1,2 = 0.004 Р1,3 = 0.004 Р2,3 = 0.000 Lymphadenitis 53.33 43.33 26.66 Р1,3 = 0.047 Р2,3 = 0.05 Sore throat 93.33 43.33 36.66 Р1,2 = 0.001 Р1,3 = 0.001 Weakness 76.66 66.66 53.33 Р1,3 = 0.001 Chills 70.00 13.33 20.00 Р1,2 = 0.001 Р1,3 = 0.001 Sweating 93.33 53.33 46.66 Р1,2 = 0.001 Р1,3 = 0.001 Runoff of mucus 33.33 13.33 16.66 Р1,2 = 0.05 Decreased concentration of attention and memory 56.66 40.00 36.66 Р1,3 = 0.050 Table 6. Frequency of main clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients of the 1st group (1st subgroup—with the level of induced IFN-γ closer to the upper limit of the reference values). Table 6. Frequency of main clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients of the 1st group (1st subgroup—with the level of induced IFN-γ closer to the upper limit of the reference values). Table 6. Frequency of main clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients of the 1st group (1st subgroup—with the level of induced IFN-γ closer to the upper limit of the reference values). subfebrile temperature, sore throat, chills, sweating, and decreased concentration. The rest of the complaints tended to decrease or remain unchanged. The dynamics of clinical complaints in the patients of the 2nd subgroup is presented in Table 7. Table 7. Frequency ofclinical complaints (%) in patientsbeforethe start,one and three months after IFN-γ therapy in patients of the 1st group (2nd subgroup—with the level of induced IFN-γ closer to the lower limit of the reference values). 3.3 Dynamics of clinical complaints Frequency of complaints (%) Before therapy (n = 21) After 1 month of therapy After 3 months of therapy p Lymphadenitis 66.66 14.28 19.04 Р1,2 = 0.002 Р1,3 = 0.05 Sore throat 33.33 23.80 19.04 Р1,3 = 0.002 Р1,3 = 0.002 Chills 47.67 28.57 23.80 Р1,2 = 0.001 Р1,3 = 0.001 Sweating 61.90 52.38 47.67 Р1,2 = 0.029 Р1,3 = 0.001 Runoff of mucus 21.05 10.52 10.52 Р1,2 = 0.029 Р1,3 = 0.029 Stomatitis 15.78 10.52 9.52 Р1,2 = 0.004 Р1,3 = 0.001 Joint pain 15.78 10.52 9.52 Р1,2 = 0.004 Р1,3 = 0.001 Decreased concentration of attention and memory 33.33 23.80 26.31 Р1,2 = 0.002 Sleep disturbance 15.78 14.28 10.52 Р1,3 = 0.004 Р2,3 = 0.046 14 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 In the 2nd subgroup of patients, one and three months after IFN-γ therapy, a significant positive dynamics of the main clinical complaints were observed, in par- ticular, a decrease in lymphadenitis, sore throat, chills, sweating, mucus drainage along the back of the throat, stomatitis, joint pain, decreased concentration attention, sleep disorders. That is, patients with an initially reduced level of induced IFN-γ before starting IFN-γ therapy have a more pronounced response to the therapy. g γ py p p py When analyzing the clinical picture, we revealed that in the group of patients with a higher level of induced IFN-γ production at the time of initiation of therapy com- plaints were more intensive and occurred with higher frequency (Figures 4 and 5). 3.5 Prognostic significance of the number of copies of EBV DNA In order to clarify the prognostic significance of the number of EBV DNA in saliva samples, a linear regression analysis was carried out with the calculation of the coefficients of determination R2 (R Square) and using the Durban-Watson test and analysis of variance (ANOVA “Analysis of Variance”) using criterion F and the calculation of the standardized coefficient beta (ß) with a 95% confidence interval. The results of the F criterion and the ß coefficient, indicating the significance of the obtained regression models, are presented below. • The number of EBV DNA copies in saliva samples before the initiation of IFN-γ therapy affects the level of induced IFN-α production one month after the end of therapy (F = 12.166; p = 0.002; β = 0.615; CI: 75.999; 264.975; p = 0.002); • The number of copies of EBV DNA in saliva samples before the initiation of IFN-γ therapy affects the level of induced IFN-γ production one month after the end of therapy (F = 3.852 p = 0.061; β = 0.365; CI:-0.011; 0.001; p = 0.061). Figure 4. Figure 4. Frequency of main clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients with CEBVI in the 1st subgroup. g Frequency of main clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients with CEBVI in the 1st subgroup. Figure 5. Frequency of clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients with CVEBI in the 2nd subgroup. Figure 5. Frequency of clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients with CVEBI in the 2nd subgroup. gu e 5. Frequency of clinical complaints (%) in patients before the start, one and three months after IFN-γ therapy in patients with CVEBI in the 2nd subgroup. 15 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control 3.4 Relationship between the number of EBV DNA copies and clinical complaints Further, a correlation analysis was carried out to reveal the relationship between the numbers of EBV DNA copies in saliva samples before the start of IFN-γ therapy with the severity of clinical complaints in patients. The results are shown in Table 8. Complaints Сorrelation coefficient Weakness τ = 0.473; р = 0.026 r = 0.553; р = 0.026 Sore throat τ = 0.629; р = 0.002 r = 0.741; р = 0.001 Joint pain τ = 0.413; р = 0.052 r = 0.521; р = 0.039 Table 8. Influence of the number of EBV DNA copies on the severity of clinical complaints in patients with СEBVI (N = 51). Table 8. Influence of the number of EBV DNA copies on the severity of clinical complaints in patients with СEBVI (N = 51). Thus, the number of copies of EBV DNA in saliva samples affects the develop- ment of weakness, sore throat, and arthralgia in patients with CEBVI. 4. Discussion Currently, there is no single approach to the treatment of CEBVI, despite the fact that there are a number of specific antiviral drugs. In particular, acyclic nucle- osides are widely used, such as acyclovir, valaciclovir (Valtrex), famciclovir (Famvir), and synthetic nucleoside analogs of guanosine: ganciclovir (cymevene), valganciclovir (Valcyte). In most cases, antiviral therapy is ineffective, which has been confirmed in numerous studies. In 2016, the results of efficacy analysis of infectious mononucleosis treatment were published according to the WHO World Register of Clinical Trials, both completed and ongoing. It was shown that the effectiveness of antiviral drugs (acyclovir, valaciclovir) in acute infectious mono- nucleosis is doubtful. Acyclovir and valaciclovir reduce EBV replication by inhibiting viral DNA polymerase and decreasing the oral secretion of EBV in patients with infectious mononucleosis. Balfour HH, et al. showed that taking the drug in a dose of 1 g every 8 h for 14 days leads to a decrease in clinical complaints in infectious mononucleosis [46]. In the case of viral shedding, shedding was observed to suppress the shedding against the background of antiviral therapy, but this effect ceased after the end of antiviral therapy [47]. The authors did not obtain a statistically significant difference between the groups of patients receiving antiviral drugs and the control groups. Most of the studies processed were unclear or at high risk of bias. Experimental studies in vitro have shown that EBV thymidine kinase has a variable affinity for antiherpetic antiviral drugs, that is, acyclovir and dihydroxypropylmethylguanine are relatively weak substrates for EBV thymidine kinase [48]. In our study, in the group of patients receiving therapy with Valtrex (valaciclovir) at a dose of 1 g per day for 2 months, suppression of EBV DNA replication in saliva samples was obtained in 28.57% of patients. This is confirmed by literature. The effectiveness of the use of valganciclovir in suppressing EBV replication and reducing the severity of clinical complaints in patients has been shown [49]. Taking valganciclovir leads to a decrease in the amount of EBV DNA from an average of 4.3 log10 copies/ml to 1.2 log10 copies/ml by 0.77 logs (95% CI, .62–.91 logs; P < .001) [50]. Valaciclovir and famciclovir suppress EBV DNA replication by 18% and 30%, respectively [51], while valganciclovir reduces EBV DNA secretion by 46% [52]. That is, valganciclovir can be used in the treatment of CEBVI. The research by Kure S. 3.6 Influence of the induced IFN-γ level on clinical complaints The next stage of the work was the correlation analysis of the influence of the initial level of induced IFN-γ on the clinical picture of the disease in patients of the 1st and 2nd subgroups (Group 1). It was shown that in the 1st subgroup, a high level of induced IFN-γ inversely influences the development of sweating in patients (r = 0.506; p = 0.023; τ = 0.419; p = 0.021). In subgroup 2, the initially low level of induced IFN-γ inversely influences the development of weakness (r = 0.405; p = 0.045; τ = 0.419; p = 0.037). It was not possible to identify other significant correlations in the subgroups. 16 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 4. Discussion et al., devoted to the study of the inhibitory effect of pure recombinant human (rh) IFN-α and IFN-γ on EBV infection in B-cell lines, BJAB lines, and in normal mature B-lymphocytes, showed that the pretreatment of cells within 24 h with rhIFN-α and rhIFN-γ suppressed the production of EBV specific nuclear antigen (EBNA-1) in BJAB cells 24 h after EBV infection. Both rhIFN types also effectively inhibited EBV infection in normal mature B lympho- cytes, as evidenced by a decrease in [3H] thymidine incorporation 6 days after EBV infection and the total number of proliferating cells 21 days after infection. The authors showed that rhIFN-α exhibited a more pronounced inhibitory effect than rhIFN-γ. None of the rhIFNs showed a pronounced inhibitory effect on EBNA expression in covert EBV-infected Raji and Daudi cell lines. These results indicate that rhIFNs act predominantly at the early stage of EBV infection [53]. In our work, it was shown that in group 1 (N = 51) one month after rhIFN-γ therapy, 15 (29.41%) patients had negative PCR results in saliva samples, and 36 (70.59%) patients had copies of EBV DNA decreased. That is, rhIFN-γ can completely inhibit viral repli- cation in 29.41% of patients. However, in this group of patients, a pronounced reliable dynamics of clinical complaints were obtained after the end of therapy. In 2002, it was shown that treatment of Vero cells with IFN-β or IFN-γ inhibits HSV-1 replication by less than 20-fold, while co-treatment with IFN-β and IFN-γ inhibits 17 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control HSV-1 replication by 1000 times [41]. The authors suggested that the high level of inhibition achieved by the administration of exogenous IFN-γ is the result of a synergistic interaction with endogenous IFN-α/β, which are locally produced in response to HSV-1 infection. Our results confirm these in vitro data. If we compare the results we obtained in the groups of patients who received rhIFN-γ and monotherapy with valganciclovir in terms of the dynamics of the number of DNA copies in saliva samples, then no difference was obtained between these groups, that is, the effectiveness of monotherapy with rhIFN-γ or valaciclovir has similar efficacy (29.41% and 28.57% respectively). 4. Discussion Our results are consistent with previ- ously published data, in particular, the Russian literature describes the results of the study of rhIFN-γ (Ingaron) and presents evidence of the high efficiency of its use in the treatment of herpesvirus infections [54]. The authors showed that the drug has a direct antiviral effect, and the clinical effect is manifested through the activation of cellular immunity, which controls the viral antigen. In group 3, a month after taking the combination therapy valganciclovir+rhIFN-γ, a negative PCR result was obtained in 19 (71.74%) patients. The effectiveness of the therapy did not depend on the combination of drugs but on the duration of the course of rhIFN-γ adminis- tration. The best result from therapy was in patients who received 20 injections of rhIFN-γ in combination with valganciclovir. It was in this group that the number of copies of EBV DNA in saliva samples was negative in 87.50% of patients. Thus, a positive result on the number of EBV DNA copies during this treatment regimen is due not so much to the total combination course, but to the amount and duration of rhIFN-γ administration. γ In 2003, an open, randomized, controlled, multicenter clinical study was conducted to study the anti-fibrotic effect of rhIFN-γ in 153 patients with chronic viral hepatitis B. RhIFN-γ was introduced i/m daily at a dose of 1 MU for three months and 1 MU every other day for the following six months. As a result, it was shown that rhIFN-γ has a pronounced anti-fibrotic effect in patients with chronic hepatitis B [55]. The effectiveness of treatment was 66% in the group of patients versus 16.2% in the control group. Later in 2011, the results of the study of rhIFN-γ monotherapy in 25 HBsAg-positive patients with stage 2-4 fibrosis who received long-term rhIFN-γ therapy were published [56]. The authors also showed that long- term therapy for nine months leads to pronounced positive dynamics of inflamma- tion and fibrosis of the liver tissue. Our results with long-term administration of rhIFN-γ confirm these data. With herpes viral infection, the secretion of cytokines is altered, modulating a strong and effective antiviral immune response against infected host cells. After primary infection, herpes viruses persist in the host organism for a long time [57]. 4. Discussion Thus, the dysregulation of the production of proinflammatory cytokines is based on the fact that virions already contain molecules that directly target the proper cytokine signal- ing. After infection of host cells and transcription of viral DNA leading to translation of viral miRs into viral peptides, other mechanisms of proper immune surveillance are targeted, including, in particular, presentation of HLA class I antigen, as well as decreased expression of NKG2D ligands [61]. the EBV protein LMP1 inhibits TNF-α and induces the production and secretion of IL-10, and the miR-BHRF1-2-5p EBV blocks the proinflammatory signaling of IL-1 [60]. Cytokine signaling is a very early response to viral infection and explains the presence of corresponding inhibitory viral factors in the tegument. Thus, the dysregulation of the production of proinflammatory cytokines is based on the fact that virions already contain molecules that directly target the proper cytokine signal- ing. After infection of host cells and transcription of viral DNA leading to translation of viral miRs into viral peptides, other mechanisms of proper immune surveillance are targeted, including, in particular, presentation of HLA class I antigen, as well as decreased expression of NKG2D ligands [61]. INF-γ plays not only an important role in modulating T-cell immunity but also, having a direct antiviral activity is used as an effective therapeutic agent in the treatment of viral infection [62]. Okano et al. conducted a study of the efficacy of therapy with recombinant IFN-γ in a patient with infectious mononucleosis and X- linked lymphoproliferative syndrome (XLP). EBV-determined nuclear antigen and EBV DNA have been found in various tissues of the patient. After therapy with recombinant IFN-γ, there was positive dynamics in the reduction of virus-infected cells and a linear increase in the content of IFN-γ in the blood serum. NK cell activity remained within normal limits throughout the course of therapy. The authors suggested that cytotoxic cells can produce endogenous IFN-γ [63]. A. Linde et al. also revealed an increase in serum IFN-γ levels 24 h (p = 0.05) and 48 h (p = 0.01) after EBV infection, subsequently, the level of IFN-γ returned to baseline values [64]. In another study, in patients with acute infectious mononucleosis, an increase in the level of serum IFN-γ was shown only during the first week from the moment of infection, later the level of IFN-γ returned to normal [65]. 4. Discussion One of the factors contributing to the persistence of herpes viruses is their ability to adopt two different modes of the life cycle: latent and lytic. After primary infection, herpes viruses pass into a latent, transcriptional-translational suppressed state, which can often be interrupted by lytic episodes. During the latency phase, tran- scripts were identified, in particular, such as microRNAs (miRs), which play a role in the mechanism of evasion of the virus from the host’s immune response, includ- ing impaired interferon signaling [58]. which can often be interrupted by lytic episodes. During the latency phase, tran- scripts were identified, in particular, such as microRNAs (miRs), which play a role in the mechanism of evasion of the virus from the host’s immune response, includ- ing impaired interferon signaling [58]. It has been shown that the early EBV protein BZLF1 can block IFN-γ production by inhibiting the downstream IFN-γ signaling pathway. Essentially, BZLF1 stops the transcription of all expressed HLA class II molecules and, therefore, the activation of T-helper cells required for the induction of an immune response, inhibits IFN-γ- induced tyrosine STAT1 phosphorylation and nuclear translocation of BZLF1, It has been shown that the early EBV protein BZLF1 can block IFN-γ production by inhibiting the downstream IFN-γ signaling pathway. Essentially, BZLF1 stops the transcription of all expressed HLA class II molecules and, therefore, the activation of T-helper cells required for the induction of an immune response, inhibits IFN-γ- induced tyrosine STAT1 phosphorylation and nuclear translocation of BZLF1, reduces the expression of the IFN-γ receptor, stimulating the mechanism, with the help of which EBV can avoid the antiviral immune response during primary infection [59]. In addition, the EBV lytic transactivator Zta suppresses the production of IFN-β, reduces the expression of the IFN-γ receptor, stimulating the mechanism, with the help of which EBV can avoid the antiviral immune response during primary infection [59]. In addition, the EBV lytic transactivator Zta suppresses the production of IFN-β, 18 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 the EBV protein LMP1 inhibits TNF-α and induces the production and secretion of IL-10, and the miR-BHRF1-2-5p EBV blocks the proinflammatory signaling of IL-1 [60]. Cytokine signaling is a very early response to viral infection and explains the presence of corresponding inhibitory viral factors in the tegument. 4. Discussion Interesting data were obtained when studying the dynamics of IFN-γ level production in patients with tuberculosis, who showed a decrease in the average IFN-γ level over time (p = 0.001), but this decrease occurred during the first 8 weeks from the start of specific therapy (p = 0.019). When comparing baseline susceptible (N = 55) and drug-resistant patients (N = 18), there was no difference in the change in IFN-γ levels over time. Since the production of IFN-γ and secretion from T cells increase in response to an increase in antigenic load and then stabilize over 24 weeks, a decrease in the concentration of IFN-γ may indicate a positive response to the therapy and play the role of monitoring the response to therapy [66]. py p y g p py Our data indicate the absence of a significant increase in the production of the induced, serum, and spontaneous level of INF-γ three months after the end of therapy with INF-γ in the general group of patients, which is fully consistent with the previously published results of other authors. But when analyzed separately in each group of patients, it was shown that in the group with an initially low level, the administration of INF-γ led to a significant increase in the level of induced INF-γ three months after the end of therapy (p = 0.027). This is probably due to the initial low level of induced INF-γ and a more pronounced response to INF-γ therapy, which manifested itself in a significant positive dynamics of the main clinical complaints. Thus, we demonstrated that the dynamics of the production of the initially low level of induced INF-γ can be a marker of the positive effect of the therapy with INF-γ. py The absence of positive dynamics of the increase in the production of induced INF-γ in the general group of patients one and three months after the end of therapy with INF-γ indicates the absence of the effect of the drug on the level of production of endogenous INF-γ, which was previously demonstrated in studies by other authors. At the same time, INF-γ has a pronounced antiviral effect, which was shown earlier, and does not cause the increase of INF-γ production to the levels that would exceed the reference values. 4. Discussion 19 Current Perspectives on Viral Disease Outbreaks - Epidemiology, Detection and Control When analyzing the clinical picture, we revealed that in the group of patients with a higher level of induced IFN-γ production at the time of initiation of therapy, complaints were more pronounced and more frequent. This is probably due to a more intensive inflammatory response in this group of patients. This conclusion is supported by previously published data that these inflammatory reactions are enhanced by the presence of IFN-γ, which dramatically increases the production of inflammatory mediators by macrophages [67]. Conflict of interest The authors declare that they have no potential conflicts of interest. 5. Conclusions 1.RhINF-γ has a pronounced antiviral effect, which is expressed in a significant decrease in the number of EBV DNA copies in patients with CEBVI. 1.RhINF-γ has a pronounced antiviral effect, which is expressed in a significant decrease in the number of EBV DNA copies in patients with CEBVI. 1.RhINF-γ has a pronounced antiviral effect, which is expressed in a significant decrease in the number of EBV DNA copies in patients with CEBVI. 2.The introduction of rhINF-γ leads to positive dynamics of the clinical picture of the disease. The most pronounced positive dynamics were found in patients with an initially low level of induced INF-γ. 2.The introduction of rhINF-γ leads to positive dynamics of the clinical picture of the disease. The most pronounced positive dynamics were found in patients with an initially low level of induced INF-γ. 2.The introduction of rhINF-γ leads to positive dynamics of the clinical picture of the disease. The most pronounced positive dynamics were found in patients with an initially low level of induced INF-γ. 3.The positive dynamics of the production of the initially low level of induced INF-γ can be a marker of the effectiveness of the therapy with rhINF-γ in patients with CEBVI. 4.The efficacy of therapy in patients with CEBVI is determined by the duration of the introduction of rhINF-γ: 500,000 IU every other day at least 20 injections shows the best result. 5.In the group of CEBVI patients with an initial level of induced IFN-γ at the lower limit of reference values, therapy with rhINF-γ leads to a significant increase in the level of induced INF-γ three months after the end of therapy. 6.Therapy with rhINF-γ one and three months after the end of treatment of patients does not cause changes in the production of INF-α to levels that would exceed the reference values in patients with CEBVI. 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This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325 Author details Irina A. Rakityanskaya1 and Tatiana S. Ryabova1,2 1 Outpatient Department of Allergology-Immunology and Clinical Transfusiology City Ambulant Department N112, St. Petersburg, Russian Federation 2 Military Medical Academy Named After S.M. Kirov Ministry of Defense of the Russian Federation, St. Petersburg, Russian Federation *Address all correspondence to: tat-akyla@inbox.ru © 2021 The Author(s). Licensee IntechOpen. 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https://openalex.org/W2944721114	https://journalofpalaeogeography.springeropen.com/track/pdf/10.1186/s42501-019-0035-5	English	null	Characteristics of Early Cretaceous wildfires in peat-forming environment, NE China	Journal of Palaeogeography/Journal of palaeogeography	2,019	cc-by	7,622	© 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. Abstract Inertinite maceral compositions in coals from the Early Cretaceous Erlian, Hailar, and Sanjiang Basins in NE China are analyzed in order to reveal palaeowildfire events and palaeoclimate variations. Although huminite is the dominant maceral group in the studied basins, the inertinite group, as a byproduct of palaeowildfires, makes up a considerable proportion. Occurrence of inertinite macerals indicates that wildfires were widespread and frequent, and supports the opinion that the Early Cretaceous was a “high-fire” interval. Inertinite contents vary from 0.2% to 85.0%, mostly within the range of 10%–45%, and a model-based calculation suggests that the atmospheric oxygen levels during the Aptian and Albian (Early Cretaceous) were around 24.7% and 25.3% respectively. Frequent fire activity during Early Cretaceous has been previously related to higher atmospheric oxygen concentrations. The inertinite reflectance, ranging from 0.58%Ro to 2.00%Ro, indicates that the palaeowildfire in the Early Cretaceous was dominated by ground fires, partially reaching-surface fires. These results further support that the Cretaceous earliest angiosperms from NE China were growing in elevated O2 conditions compared to the present day. Keywords: Inertinite, Coal, Wildfire, Palaeo-atmospheric oxygen level, Angiosperm, Early Cretaceous, NE China 2004; Bowman et al. 2009). The minimum oxygen content that can support combustion in nature is 15% (Belcher and McElwain 2008). Wildfire plays an important role in many ecosystems, which not only affects the emission of atmospheric carbon dioxide and aerosols, but also plays an important controlling role in the distribution and evo- lution of the biological community (Scott and Stea 2002; Bowman et al. 2009; Scott 2010). The inertinite content in coal or other sediments is related to atmospheric oxygen levels, and thus can be used in interpretation of the palaeoenvironment of peatlands (Scott and Glasspool 2006; Diessel 2010; Glasspool and Scott 2010; Zhan et al. 2011; Shao et al. 2012; Glasspool et al. 2015). Journal of Palaeogeography Journal of Palaeogeography (2019) 8:17 Wang et al. Journal of Palaeogeography (2019) 8 https://doi.org/10.1186/s42501-019-0035-5 Open Access Characteristics of Early Cretaceous wildfires in peat-forming environment, NE China Shuai Wang, Long-Yi Shao, Zhi-Ming Yan, Ming-Jian Shi and Yun-He Zhang Correspondence: shaol@cumtb.edu.cn College of Geoscience and Surveying Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China 1 Introduction Although many studies have discussed the coal-forming environment and coal petrology of the Early Cretaceous coals of NE China, few studies focus on the palaeoclimate significance based on the fire-derived inertinites (Xu 2011; Zhang et al. 2014). Therefore, in this study, Early Cretaceous coals in the Erlian, Hailar and Sanjiang Basins of NE China are selected to assess the characteristics of wildfire types and atmospheric oxygen levels. The work provides further insight into the occurrences of wildfires and palaeoenvironmental changes during this period. experienced two stages of tectonic evolution under differ- ent tectonic regimes. During the Paleozoic, tectonic devel- opment was controlled by the evolution of the Palaeo- Asian Ocean between Siberia and North China cratons, but since Jurassic, it was dominated by the subduction of Palaeo-Asian Ocean and the collision of Mongolia– Okhotsk suture (Wang et al. 2007; Wu et al. 2011). The latter tectonic regime fundamentally changed the tectonic framework of NE China and initiated NE- to NNE-ori- ented tectonic zones (Li 1988), which resulted in the de- velopment of a series of Late Jurassic to Early Cretaceous NE- to NNE-oriented faulted coal-bearing basins. NE China includes Heilongjiang, Jilin and Liaoning Provinces and the eastern part of Inner Mongolia Au- tonomous Region with a vast area of roughly 1.6 million km2 (Shao et al. 2013). Tectonically, the region consists of Hailar Basin and Erlian Basin in the west, Songliao Basin in the center, and Sanjiang Basin in the east (Fig. 1), respectively. The faulted basins in NE China are 1 Introduction As a byproduct of peatland evolution under the common influence of geological conditions including palaeoclimate, palaeobotany, palaeogeography and palaeotectonics, coal is not only an important energy resource, but also a sediment sensitive to environmental changes in deep geological time (Shao et al. 2012). Therefore, the maceral compositions of coal can record subtle changes in depositional conditions and palaeoclimate (Petersen and Ratanasthien 2011; Petersen and Lindström 2012). Almost all coal macerals in the inertinite group have been identified as incompletely combusted charcoal and are closely tied to wildfires in peatlands (Scott 2000, 2010; Scott and Stea 2002; Scott and Glasspool 2006, 2007; Glasspool and Scott 2010; Hudspith et al. 2012; Zhang et al. 2016). The occurrence of wildfire needs sufficient temperature, fuel and oxygen conditions (Brown et al. 2012). Charcoal, as the byproduct of wildfire, is first re- corded in the latest Silurian and has subsequently been found in all geological periods from different depositional environments (Jones and Chaloner 1991; Glasspool et al. The Cretaceous is an important coal-forming period in geological history (Han and Yang 1980) and is character- ized by global greenhouse climates (Hu et al. 2012; Wang 2013). Coal seams of this period are widely distributed around the world (Brown et al. 2012). Previous research on wildfires in coal mainly investigated coals in the northern hemisphere (North America, Europe, India, and Asia) (Uhl and Kerp 2003; Hamad et al. 2016; Jasper et al. 2017). Wildfires were widespread and frequent during large parts of the Cretaceous, and thus the Cretaceous has been con- sidered as a high frequency interval of wildfires in Earth’s * Correspondence: shaol@cumtb.edu.cn College of Geoscience and Surveying Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China * Correspondence: shaol@cumtb.edu.cn College of Geoscience and Surveying Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China Page 2 of 13 Page 2 of 13 Wang et al. Journal of Palaeogeography (2019) 8:17 Wang et al. Journal of Palaeogeography history (Bond and Scott 2010; Diessel 2010; Brown et al. 2012). It is believed that the frequent occurrence of fire events is related to the abundance of gymnosperms and the emergence of angiosperms (Bond and Scott 2010; Brown et al. 2012). 4.1 Maceral compositions Table 1 shows the maceral and mineral matter distributions of the samples collected from the three basins studied. In the Erlian Basin, huminite is the most dominant mac- eral group (14.4%–99.3%) with an average value of 63.1%, but a few samples exhibit higher values of inertinite and lip- tinite contents as well (Table 1). The inertinite content of all samples varies between 0.2% and 85.0% with an average value of 33.9%. Liptinite is not commonly observed, the content of which ranges between 0.2% and 15.0% with an average value of 3.0%. Fusinite, semifusinite and iner- todetrinite are the abundant inertinite macerals with average content at 13.4%, 8.1%, 11.3%, respectively. Funginite, macrinite and micrinite are rarely found in the samples. The minerals comprise 0.8%–17.7% with an average value of 4.9%. From bottom to top, the lithostratigraphic units of the Lower Cretaceous coal measures include the Aershan, Tengger and Saihantala Formations in the Erlian Basin; Tongbomiao, Nantun, Damoguaihe and Yimin Forma- tions in the Hailar Basin; and Didao, Chengzihe and Muling Formations in the Sanjiang Basin, respectively (Fig. 2). Chronostratigraphically, they correspond to the Aptian and most of the Albian, Early Cretaceous (Sun and Zheng 2000; Zhang et al. 2015; ICS 2017) (Fig. 2). In the Hailar Basin, it is noted that huminite and iner- tinite are the dominant maceral groups, followed by mineral components, while liptinite is rare (Table 1). The huminite group is 81.5% in average of the total mac- erals, ranging from 40.2% to 95.6%, the inertinite group 18.6% in average, ranging from 4.4% to 59.8%, and the mineral components are 1.3% in average. Fusinite and semifusinite are the abundant inertinite macerals with average content at 17.8% and 0.7%, respectively. 3 Sampling and methodology p g gy The studied coal samples were collected from: (1) coal mines in the Saihantala Formation of the Jiergalangtu, Baiyinhua, and Chaokewula sags in the Erlian Basin; (2) the Yimin Formation of the Yimin sag in the Hailar Basin; and (3) the Chengzihe Formation of Jixi mine area in the Sanjiang Basin (Fig. 3), comprising 19, 12, and 5 samples, respectively. All the collected samples were stored imme- diately in plastic bags to ensure as little contamination or oxidation as possible. Samples were analyzed in the State Key Laboratory of Coal Resources and Safe Mining, Beijing, China. The maceral content was determined by point counting, and huminite and inertinite reflectance measurements were carried out on most samples. The coal petrographic analyses were made according to con- ventional procedures (ICCP 2001; Sýkorová et al. 2005). In the Sanjiang Basin, the huminite is also the domin- ant group, ranging from 74.9% to 92.0%, with an average value of 87%, while the inertinite group accounts for 9.8% in average, ranging from 2.7% to 24.3% (Table 1). The liptinite is 3.2% in average and the mineral compo- nents are 0.4% in average. Fusinite and semifusinite are the major inertinite macerals with average content at 6.2% and 3.6%, respectively. 2 Geological setting Northeastern China is located at the eastern part of the Central Asian Orogenic Belt (Wu et al. 2011), which Fig. 1 Outline map showing the Early Cretaceous sedimentary basins of NE China (modified from Sha 2007). The inset map of China is modified after the National Bureau of Surveying and Mapping of China (No. GS(2016)1603). I – Hailar Basin; II – Erlian Basin; III – Sanjiang Basin Fig. 1 Outline map showing the Early Cretaceous sedimentary basins of NE China (modified from Sha 2007). The inset map of China is modified after the National Bureau of Surveying and Mapping of China (No. GS(2016)1603). I – Hailar Basin; II – Erlian Basin; III – Sanjiang Basin Page 3 of 13 Wang et al. Journal of Palaeogeography (2019) 8:17 Page 3 of 13 Wang et al. Journal of Palaeogeography (0.590%Ro), and optically black (zero) standards at 23 °C were used, and a total of 100 valid points in each polished block were counted. Finally, the testing results are con- verted into percentage of each maceral and average re- flectance value in the total valid points. For observation of the homogenization status of cell walls, macerals were ob- served under scanning electron microscopy. filled by Lower Cretaceous coal measures, mostly continen- tal clastics, including conglomerate, sandstone, siltstone, mudstone and coal, from 1000 m to 3000 m in thickness (Li 1988), formed mainly in continental environments. Lower Cretaceous successions are unconformably overlain by Upper Cretaceous sedimentary rocks, and unconform- ably underlain by the Upper Jurassic (Shao et al. 2013). The Hailar Basin consists of up to 6 km of Upper Mesozoic–Cenozoic continental sediments and forms a NE-trending basin together with the adjacent Tamtsag Basin in Mongolia to the south (Gao et al. 2009; Guo et al. 2018). The Erlian Basin is a Mesozoic–Cenozoic intra-continental faulted basin, developed on Hercynian folded basement, which is located in the suture between the Asian and Siberian Plates (Zhu et al. 2000). The San- jiang Basin is an important Mesozoic coal-bearing basin, which is located in the Jiamusi Massif of the eastern Heilongjiang Province (Yang et al. 2005). 4.2 Inertinite reflectance values Coal samples were crushed to less than 1 mm diameter to make polished blocks for microscopic observation. Maceral analyses, as well as huminite and inertinite re- flectance measurements were made on polished blocks under reflected white and fluorescent light, with 50× oil immersion objectives using a Leica DM4500P LED micro- scope. For the maceral analyses, a total of 500 valid points in each polished block were counted. For the reflectance measurements, an immersion oil with a refractive index of 1.518 and YAG (0.903%Ro), GGG (1.719%Ro), sapphire Since inertinite reflectance is affected by combustion temperature, the temperature of wildfire combustion can be inferred by measuring the inertinite reflectance (Petersen and Lindström 2012). Although there is no completely linear relationship between inertinite reflect- ance and burning temperature, it can be described by the linear regression eq. T = 184.10 + 117.76 × %Ro (coef- ficient of determination r2 = 0.91), where T is the com- bustion temperature and %Ro is the measured inertinite reflectance (Jones 1997). Page 4 of 13 Wang et al. Journal of Palaeogeography (2019) 8:17 Wang et al. Journal of Palaeogeography Fig. 2 Columnar sections showing lithology and coal seams of the Lower Cretaceous coal measures in the Erlian, Hailar and Sanjiang Basins. The palynological data are based on Heilongjiang Bureau of Geology and Mineral Resources (1993), Zhang and Long (1995) and Zhu et al. (2000) Fig. 2 Columnar sections showing lithology and coal seams of the Lower Cretaceous coal measures in the Erlian, Hailar and Sanjiang Basins. The palynological data are based on Heilongjiang Bureau of Geology and Mineral Resources (1993), Zhang and Long (1995) and Zhu et al. (2000) Sanjiang Basin is 294.79–330.12 °C with an average value of 308.22 °C. The measured inertinite reflectance results are shown in Table 2, from which it can be seen that inertinite re- flectance in the Erlian Basin is 0.59%Ro–2.00%Ro with an average of 1.03%Ro; inertinite reflectance in the Hai- lar Basin is 0.58%Ro–0.90%Ro, with an average value of 0.75%Ro; and the Sanjiang Basin inertinite reflectance of 0.94%Ro–1.24%Ro, with an average value of 1.05%Ro. Based on the relationship between inertinite reflectance and combustion temperature (Jones 1997), the wildfire combustion temperature for the studied sections in NE China was calculated (Table 2). 4.2 Inertinite reflectance values The combustion temperature of wildfire in the Erlian Basin is 253.58– 419.62 °C with an average value of 305.34 °C; the wildfire combustion temperature in the Hailar Basin is 252.40–290.08 °C with an average value of 271.83 °C; and the wildfire combustion temperature in the 5.1 Inertinite macerals: wildfire implications 5.1 Inertinite macerals: wildfire implications A mix of inertinite particle sizes from more than 500 μm to less than 10 μm could be observed in polished blocks, which recorded local wildfire activity and regional activ- ity (Scott and Glasspool 2007; Robson et al. 2015). The common characteristics of inertinite are the relatively high reflectivity (Fig. 3). Fusinite, semifusinite, and iner- todetrinite are the most common inertinite macerals (Fig. 3a–c, e). Fusinite and semifusinite have open cellu- lar structure and homogenized cell walls (Fig. 4). Com- paratively, semifusinite has lower reflectivity than fusinite (Fig. 3e). Rare inertinite in areas indicates that Page 5 of 13 Wang et al. Journal of Palaeogeography (2019) 8:17 (2019) 8:17 Wang et al. Journal of Palaeogeography Fig. 3 Photomicrographs of typical coal macerals in the studied coals. Inertinite shows higher reflectivity than huminite. Huminite maceral group: ulminite and corpohuminite. Inertinite maceral group: fusinite, semifusinite and Inertodetrinite. Oil immersion, white light. a, b Distinct cellular structure is preserved in the fusinite (Fu); from sample 407, Hailar Basin; c, d Fusinite (Fu) with distinct cellular structure and corpohuminite (Co) with spherical or oval shape indicating resistance to decay; from sample 419, Hailar Basin; e Semifusinite (Sf) with brittle fractures has contacts with ulminite (Ul); from sample 302, Sanjiang Basin; f Due to homogenization, structures within the cell walls are not visible in the ulminite (Ul); from sample 211, Erlian Basin Fig. 3 Photomicrographs of typical coal macerals in the studied coals. Inertinite shows higher reflectivity than huminite. Huminite maceral group: ulminite and corpohuminite. Inertinite maceral group: fusinite, semifusinite and Inertodetrinite. Oil immersion, white light. a, b Distinct cellular structure is preserved in the fusinite (Fu); from sample 407, Hailar Basin; c, d Fusinite (Fu) with distinct cellular structure and corpohuminite (Co) with spherical or oval shape indicating resistance to decay; from sample 419, Hailar Basin; e Semifusinite (Sf) with brittle fractures has contacts with ulminite (Ul); from sample 302, Sanjiang Basin; f Due to homogenization, structures within the cell walls are not visible in the ulminite (Ul); from sample 211, Erlian Basin Hailar Basin) to lower wildfire activity (Sanjiang Basin). fuel would have accumulated between periods of fire ac- tivity (Robson et al. 2015). Enrichment of inertinite in Lower Cretaceous coals is very common in the studied area. The coals in the western part of the study area contain higher charcoal abundances than those in the eastern part based on the coal petrological evidence (Table 1). Higher levels of inertinite in contemporaneous coals have also been recorded from New Zealand, America, Canada, Africa, and North China (Diessel 2010; Sun et al. 2017). The variability in inertinite content indicates that wildfires were prevalent in the coal-forming peatland during the Early Cretaceous, and may record transitional periods from higher wildfire activity (Erlian Basin and 5.2 Fire types According to different temperature and fuel, wildfires can be subdivided into three categories (Scott 1989; Scott and Jones 1994; Scott 2000), namely: (1) ground fires that burn organic material below litter, producing temperatures of around 400 °C; (2) surface fires that burn litter and herbaceous and shrubby plants, produ- cing temperatures of around 600 °C; and (3) crown fire that burn the canopy of trees and lager shrubs, produ- cing temperatures of 800 °C or higher. Based on the calculated burning temperature, we have summarized Wang et al. Journal of Palaeogeography (2019) 8:17 Page 6 of 13 Table 1 Maceral and mineral compositions of the Lower Cretaceous coals in NE China Area Sample No. 5.2 Fire types Total macerals (vol.%) Total minerals (vol.%) Percentage of maceral groups in the total macerals (vol.%, mmf) Percentage of inertinite macerals in the total macerals (vol.%, mmf) Huminite Reflectance (%Ro) Huminite Liptinite Inertinite Fusinite Semifusinite Sclerotinite Macrinite Micrinite Inertodetrinite Hailar Basin 401 100.0 0.0 84.7 0.0 15.3 14.6 0.7 0.0 0.0 0.0 0.0 0.42 407 99.5 0.5 84.2 0.0 15.8 15.8 0.0 0.0 0.0 0.0 0.0 0.38 411 99.8 0.2 79.4 0.0 20.6 18.2 2.4 0.0 0.0 0.0 0.0 0.43 416 93.8 6.2 93.6 0.0 6.4 4.7 1.7 0.0 0.0 0.0 0.0 0.40 419 97.4 2.6 87.2 0.0 12.8 12.8 0.0 0.0 0.0 0.0 0.0 0.42 426 98.3 1.7 89.2 0.0 10.8 10.8 0.0 0.0 0.0 0.0 0.0 0.40 431 98.8 1.2 90.9 0.0 9.1 9.1 0.0 0.0 0.0 0.0 0.0 0.39 436 98.5 1.5 83.8 0.0 16.2 16.2 0.0 0.0 0.0 0.0 0.0 0.35 441 100.0 0.0 81.4 0.0 18.6 18.6 0.0 0.0 0.0 0.0 0.0 0.40 446 99.6 0.4 95.6 0.0 4.4 4.4 0.0 0.0 0.0 0.0 0.0 0.30 452 99.4 0.6 67.2 0.0 32.8 32.8 0.0 0.0 0.0 0.0 0.0 0.32 455 100.0 0.0 40.2 0.0 59.8 55.8 4.0 0.0 0.0 0.0 0.0 0.41 Sanjiang Basin 302 100.0 0.0 92.0 2.3 5.7 3.5 2.2 0.0 0.0 0.0 0.0 0.55 305 100.0 0.0 88.7 1.5 9.8 6.8 3.0 0.0 0.0 0.0 0.0 0.41 310 99.8 0.2 74.9 0.8 24.3 19.2 5.1 0.0 0.0 0.0 0.0 0.57 311 99.3 0.7 91.5 2.2 6.3 1.5 4.8 0.0 0.0 0.0 0.0 0.60 313 99.0 1.0 88.1 9.2 2.7 0.0 2.7 0.0 0.0 0.0 0.0 0.44 Erlian Basin 201 99.2 0.8 64.2 1.2 34.6 6.5 15.0 0.0 0.0 0.0 13.1 0.32 203 98.1 1.9 68.7 5.4 25.9 4.9 4.2 0.0 1.2 0.2 15.4 0.40 204 95.2 4.8 86.3 2.3 11.3 5.0 2.7 0.0 0.0 0.0 3.6 0.33 205 91.0 9.0 89.2 1.5 9.2 1.3 2.7 0.0 0.0 0.0 5.2 / 206 96.3 3.7 96.8 1.9 1.3 0.4 0.0 0.0 0.0 0.0 0.9 0.33 207 94.2 5.8 98.7 0.6 0.6 0.2 0.0 0.0 0.0 0.0 0.4 / 208 92.0 8.0 29.3 9.5 61.2 20.4 19.7 0.2 5.0 0.0 15.9 0.42 209 94.4 5.6 54.3 1.4 44.3 16.6 11.2 0.0 0.2 0.0 16.3 / 210 98.0 2.0 31.1 2.2 66.7 33.1 17.9 0.6 0.6 0.0 14.5 0.39 211 96.9 3.1 67.5 1.7 30.9 18.0 2.1 0.0 0.0 0.0 10.8 / 212 94.6 5.4 14.4 0.6 85.0 39.5 11.9 0.0 0.2 0.0 33.4 / 213 97.6 2.4 34.2 2.0 63.7 29.6 9.9 0.0 0.4 0.2 23.6 / 214 98.0 2.0 58.8 6.1 35.1 7.4 14.9 0.0 1.0 0.0 11.8 0.34 215 97.4 2.6 63.1 9.4 27.4 7.3 8.2 0.2 0.6 0.0 11.1 / Page 7 of 13 Wang et al. 5.2 Fire types Journal of Palaeogeography (2019) 8:17 Wang et al. Journal of Palaeogeography (2019) 8:17 ble 1 Maceral and mineral compositions of the Lower Cretaceous coals in NE China (Continued) ea Sample No. Total macerals (vol.%) Total minerals (vol.%) Percentage of maceral groups in the total macerals (vol.%, mmf) Percentage of inertinite macerals in the total macerals (vol.%, mmf) Huminite Reflectance (%Ro) Huminite Liptinite Inertinite Fusinite Semifusinite Sclerotinite Macrinite Micrinite Inertodetrinite 216 89.0 11.0 95.1 3.9 1.0 0.2 0.0 0.0 0.2 0.0 0.6 0.32 217 94.1 5.9 29.9 1.1 69.0 44.4 6.8 0.2 0.4 0.0 17.2 / 218 97.2 2.8 83.7 15.0 1.2 0.2 0.0 0.0 0.0 0.0 1.0 / 219 82.3 17.7 99.3 0.5 0.2 0.0 0.0 0.0 0.0 0.0 0.2 / 220 98.8 1.2 32.1 0.4 67.5 19.9 27.2 0.0 0.2 0.0 20.2 0.36 14–1* 98.6 1.4 68.4 0.2 31.4 22.8 3.7 0.7 0.2 0.0 4.0 / 14–2* 97.6 2.4 76.2 1.4 22.4 8.1 3.6 0.4 0.0 0.0 10.3 / 14–3* 98.8 1.2 71.5 1.8 26.7 11.5 3.5 1.0 0.2 0.0 10.5 / 11–1* 99.1 0.9 14.9 0.5 84.6 54.2 6.1 0.2 1.9 0.0 22.2 / 11–2* 84.5 15.5 86.9 1.5 11.6 2.2 0.4 0.4 0.0 0.0 8.6 / ata from the Tengger Formation of the Huolinhe sag in the Erlian Basin (Xu 2011). mmf Mineral-matter-free (= no mineral matter) Wang et al. Journal of Palaeogeography (2019) 8:17 Page 8 of 13 Table 2 Measured inertinite reflectance and inferred burning temperatures of wildfire in the Lower Cretaceous coals, NE China Area Sample No. Data from the Tengger Formation of Huolinhe sag in Erlian Basin (Xu 2011) T = 184.10 + 117.76 × %Ro (Jones 1997) 5.2 Fire types Effective points Measured inertinite reflectance (%Ro) Inferred burning temperature T* (°C) Minimum Maximum Average Hailar Basin 401 100 0.42 2.06 0.88 287.73 407 101 0.51 1.60 0.77 274.78 411 100 0.51 2.07 0.80 278.31 416 101 0.44 1.43 0.90 290.08 419 107 0.22 1.82 0.72 268.89 426 101 0.47 0.99 0.73 270.06 431 100 0.43 1.25 0.67 263.00 436 100 0.37 1.30 0.68 264.18 441 101 0.36 1.65 0.73 270.06 446 70 0.34 1.44 0.72 268.89 452 101 0.28 1.04 0.58 252.40 455 100 0.52 1.17 0.76 273.60 Sanjiang Basin 302 103 0.63 2.05 1.01 303.04 305 101 0.54 1.52 1.04 306.57 310 100 0.59 2.07 1.24 330.12 311 100 0.60 1.90 0.94 294.79 313 101 0.70 2.06 1.04 306.57 Erlian Basin 201 105 0.34 1.00 0.64 259.47 203 97 0.62 1.88 1.02 304.22 204 102 0.28 2.08 1.05 307.75 205 102 0.38 1.67 0.86 285.37 206 101 0.42 2.06 1.03 305.39 207 100 0.40 2.07 1.05 307.75 208 101 0.59 2.07 1.06 308.93 209 100 0.63 2.06 1.22 327.77 210 105 0.44 1.36 0.71 267.71 211 103 0.31 1.17 0.68 264.18 212 100 0.57 1.36 0.88 287.73 213 100 0.43 2.06 0.93 293.62 214 101 0.43 1.86 0.83 281.84 215 101 0.53 1.73 0.78 275.95 216 104 0.36 1.95 0.80 278.31 217 101 0.53 1.64 0.99 300.68 218 101 0.38 1.58 0.65 260.64 219 67 0.40 1.01 0.59 253.58 220 102 0.61 2.07 1.24 330.12 14–1* / / 2.10 1.30 337.19 14–2* / / 2.80 2.00 419.62 14–3* / / 2.90 1.30 337.19 11–1* / / 3.00 2.00 419.62 11–2* / / 1.20 1.10 313.64 Data from the Tengger Formation of Huolinhe sag in Erlian Basin (Xu 2011) *T = 184.10 + 117.76 × %Ro (Jones 1997) Page 9 of 13 Wang et al. Journal of Palaeogeography (2019) 8:17 (2019) 8:17 Wang et al. Journal of Palaeogeography Fig. 4 Scanning electron microscope micrographs of inertinite in the studied coals. Fusinite, one of the major maceral types in the inertinite group, shows structured and homogenized cell walls, indicating a wildfire origin. a Longitudinal section of fusinite shows fibrous structure. Sample 416, Yimin Formation, Hailar Basin; b Longitudinal section of fusinite shows fibrous structure and pitting. Sample 441, Yimin Formation, Hailar Basin; c Cross section of fusinite shows weakly deformed cellular pore in fusinite. Sample 455, Yimin Formation, Hailar Basin; d Cross section of fusinite shows deformed cellular pores in fusinite. 5.2 Fire types Sample 455, Yimin Formation, Hailar Basin Fig. 4 Scanning electron microscope micrographs of inertinite in the studied coals. Fusinite, one of the major maceral types in the inertinite group, shows structured and homogenized cell walls, indicating a wildfire origin. a Longitudinal section of fusinite shows fibrous structure. Sample 416, Yimin Formation, Hailar Basin; b Longitudinal section of fusinite shows fibrous structure and pitting. Sample 441, Yimin Formation, Hailar Basin; c Cross section of fusinite shows weakly deformed cellular pore in fusinite. Sample 455, Yimin Formation, Hailar Basin; d Cross section of fusinite shows deformed cellular pores in fusinite. Sample 455, Yimin Formation, Hailar Basin 5.3 Fires in the Early Cretaceous peatland and the implications for atmospheric oxygen levels the fire types during the Aptian and Albian. During the Aptian, most inertinite has a low reflectance (0.94%– 1.30%) and burning temperature (294.79–337.19 °C), in- dicating that they are mainly derived from ground fire. Some inertinite has high-reflectance values suggesting that it was derived from high temperatures, partially reaching surface fires (Fig. 5b). Sporo-pollens in coals show that the vegetation in peatland was dominated by conifers (gymnosperms) (Xu 2011). During the Albian, all inertinite macerals have low reflectance (0.58%– 1.24%) and low burning temperature (252.40–330.12 °C), suggesting they were mainly derived from ground fire (Fig. 5a). Sporo-pollen assemblages in the coals indicate that the vegetation was dominated by conifers and ferns (pteridophytes) (Shen et al. 2018), which might feed the ground fire. the fire types during the Aptian and Albian. During the Aptian, most inertinite has a low reflectance (0.94%– 1.30%) and burning temperature (294.79–337.19 °C), in- dicating that they are mainly derived from ground fire. Some inertinite has high-reflectance values suggesting that it was derived from high temperatures, partially reaching surface fires (Fig. 5b). Sporo-pollens in coals show that the vegetation in peatland was dominated by conifers (gymnosperms) (Xu 2011). During the Albian, all inertinite macerals have low reflectance (0.58%– 1.24%) and low burning temperature (252.40–330.12 °C), suggesting they were mainly derived from ground fire (Fig. 5a). Sporo-pollen assemblages in the coals indicate that the vegetation was dominated by conifers and ferns (pteridophytes) (Shen et al. 2018), which might feed the ground fire. The wildfire activity can be almost influenced by the changes in lighting, fuel type, temperature and moisture (Scott 2000; Belcher et al. 2010; Robson et al. 2015). 5.2 Fire types Global climate patterns from the Early Cretaceous show a long-term greenhouse trend (Hu et al. 2012). During the Early Cretaceous, global warming associated with in- creased atmospheric carbon dioxide levels is likely to have enhanced storm tracks and convective storms, leading to a likely increase in the frequency of lightning strikes and subsequent ignition of wildfires (Belcher et al. 2010). The seasonality and intensity of precipitation affect groundwater level and moisture content of fuel, deter- mining fire ignition and spread in peat swamp ecosys- tems (Usup et al. 2004; Yan et al. 2016). Fire combustion Page 10 of 13 Wang et al. Journal of Palaeogeography (2019) 8:17 Wang et al. Journal of Palaeogeography Fig. 5 Inertinite reflectance histograms showing calculated burning temperature (T = 184.10 + 117.76 × %Ro) of selected reflectance intervals and the possible types. a Sample from the Saihantala and Yimin Formations corresponding to the Albian. All inertinite has low reflectance and low temperature suggesting that it was derived from ground fire; b Sample from the Tengger and Chengzihe Formations of the Aptian. 80% inertinite of low reflectance and low temperature is interpreted to indicate that it was derived from ground fire, while 20% inertinite of high reflectance suggests that it was derived from high temperatures reached in surface fires. n Number of samples Fig. 5 Inertinite reflectance histograms showing calculated burning temperature (T = 184.10 + 117.76 × %Ro) of selected reflectance intervals and the possible types. a Sample from the Saihantala and Yimin Formations corresponding to the Albian. All inertinite has low reflectance and low temperature suggesting that it was derived from ground fire; b Sample from the Tengger and Chengzihe Formations of the Aptian. 80% inertinite of low reflectance and low temperature is interpreted to indicate that it was derived from ground fire, while 20% inertinite of high reflectance suggests that it was derived from high temperatures reached in surface fires. n Number of samples Fig. 5 Inertinite reflectance histograms showing calculated burning temperature (T = 184.10 + 117.76 × %Ro) of selected reflectance intervals and the possible types. a Sample from the Saihantala and Yimin Formations corresponding to the Albian. All inertinite has low reflectance and low temperature suggesting that it was derived from ground fire; b Sample from the Tengger and Chengzihe Formations of the Aptian. 5.2 Fire types 80% inertinite of low reflectance and low temperature is interpreted to indicate that it was derived from ground fire, while 20% inertinite of high reflectance suggests that it was derived from high temperatures reached in surface fires. n Number of samples suggests low moisture content of fuels, which further in- dicates a low ground water level and lower air humidity (Yan et al. 2016). Increased rainfall will result in wetter fuel and be beneficial to plant growth. When coupled with increased temperatures and higher atmospheric oxygen levels, wetter plants will have more potential to burn, although they are previously difficult to ignite (Robson et al. 2015). 25.3% respectively, which exceeded the present atmos- pheric oxygen level of 21%. Therefore, the records of iner- tinite, the large amounts of charred plant and the inferred high atmospheric oxygen levels suggest the Early Cretaceous was a high-fire period in NE China. The Early Cretaceous in NE China is an important period for the early evolution of angiosperms (Tao et al. 2013), so these results provide new information to provide context for this period of botanical innovation. In short, the earliest angio- sperms were growing in elevated O2 conditions compared to the present day. In addition to these factors, O2 is a key factor affecting the occurrence of wildfire. Based on the correlation model between inertinite content and atmospheric oxygen con- centration (p(O2)) (Glasspool et al. 2015), we have summa- rized the inertinite contents in the Aptian and Albian (about 22.6% and 27.7%, respectively), and then employed them to the established nonlinear fitting chart (Fig. 6). The result shows that p(O2) levels of the Aptian and Albian during the Early Cretaceous in NE China are 24.7% and In addition to regional effects (Glasspool and Scott 2010), wildfires activity may also provide feedback towards promoting plant diversification and increasing greenhouse gas by releasing CO2 through combustion of biomass (Chanton et al. 2000; Bond and Scott 2010). Wildfire can lead to plant mortality, and the ability of a Wang et al. Journal of Palaeogeography (2019) 8:17 Page 11 of 13 Fig. 6 Calculation of oxygen levels from the studied areas based on the model proposed by Glasspool et al. (2015). The oxygen curves have been calculated from the inertinite in coal data from Glasspool and Scott (2010) and Glasspool et al. (2015). Abbreviations C C h i Co: Corpohuminite; Fu: Fusinite; GGG: Gadolinium galium garnet; Co: Corpohuminite; Fu: Fusinite; GGG: Gadolinium galium garnet; Id: Inertodetrinite; mmf: Mineral-matter-free (= no mineral matter); Ro: Reflectance in oil; Sf: Semifusinite; T: Temperature; Ul: Ulminite; YAG: Yttrium alumimium garnet Acknowledgements 1) Lower Cretaceous coals from NE China have been studied for their maceral compositions and inertinite reflectance. The results show that although macerals in the studied coals are generally dominated by huminite, inertinite contents are significant, ranging from 0.2% to 85.0%, mostly within the range between 10% and 45%. We thank Jin-Yun Fan and Yu-Guo Kang for their assistance in sampling. We also thank the editor-in-chief, Zeng-Zhao Feng, editor Xiu-Fang Hu, reviewers Jason Hilton and Dieter Uhl for their constructive comments to improve the manuscript. 5.2 Fire types S-shaped curves are assumed to ensure smooth transition from 0% inertinite to 100% inertinite of total macerals. Three curves represent the minimum, best and maximum estimate from left to right, respectively Fig. 6 Calculation of oxygen levels from the studied areas based on the model proposed by Glasspool et al. (2015). The oxygen curves have been calculated from the inertinite in coal data from Glasspool and Scott (2010) and Glasspool et al. (2015). S-shaped curves are assumed to ensure smooth transition from 0% inertinite to 100% inertinite of total macerals. Three curves represent the minimum, best and maximum estimate from left to right, respectively indicating that wildfires were widely distributed in the Early Cretaceous coal-forming peatland. plant to survive fire obviously determines the develop- ment of plant living in disturbed environments in a high-fire world (Brown et al. 2012; Shao et al. 2012). If frequent, fire may kill saplings of larger trees, conse- quently the herbaceous vegetation can be maintained (Bond and Keeley 2005). The herbaceous plant-fire cycle shows a competitive advantage during times of warmth and high CO2 and O2 (Bond and Scott 2010). Post-fire erosion is also an important factor having a major impact on Cretaceous terrestrial environments and may add phosphorous runoff into oceanic or lacustrine settings leading to anoxia and carbon burial (Brown et al. 2012). Therefore, the relationships between Lower Cretaceous charcoal assemblages, plant evolution, oxygen levels and anoxia events in Earth system should be further studied in order to reveal the palaeoenviron- mental and palaeoecological information in the Lower Cretaceous coal-bearing strata. 3) Based on the relationship between burning temperature and inertinite reflectance, the burning temperature of palaeowildfire ranges from 252.40 °C to 419.62 °C, suggesting that the palaeowildfire in the Aptian and Albian was dominated by ground fire. 3) Based on the relationship between burning temperature and inertinite reflectance, the burning temperature of palaeowildfire ranges from 252.40 °C to 419.62 °C, suggesting that the palaeowildfire in the Aptian and Albian was dominated by ground fire. 4) Based on the established relationship between the palaeo-atmospheric O2 level and the inertinite content, the atmospheric oxygen contents of the Aptian and Albian were around 24.7% and 25.3% respectively. 5) These results support that the Cretaceous earliest angiosperms from NE China were growing in elevated O2 conditions compared to the present day. 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https://openalex.org/W1981551851	https://europepmc.org/articles/pmc4394691?pdf=render	English	null	Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via α4β2* nicotinic receptor activation	Frontiers in cellular neuroscience	2,015	cc-by	9,308	Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via α4β2* nicotinic receptor activation L. Andrew Bell , Karen A. Bell and A. Rory McQuiston * Acetylcholine (ACh) release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the speciﬁc interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF- tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP)-expressing interneurons that selectively innervate other interneurons (VIP/IS) were excited by ACh through the activation of nicotinic receptors containing α4 and β2 subunits (α4β2). ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC) barrages blocked by dihydro-β-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by α4β2 nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of α4β2* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation. Citation: Keywords: optogenetics, nicotinic receptor, CA1, hippocampus, interneuron-selective interneuron, disinhibition Bell LA, Bell KA and McQuiston AR (2015) Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via α4β2 nicotinic receptor activation. Front. Cell. Neurosci. 9:115. doi: 10.3389/fncel.2015.00115 Edited by: Tycho M. Hoogland, Erasmus MC, Netherlands Reviewed by: William Lytton, SUNY Downstate, USA Huib Mansvelder, VU University, Netherlands *Correspondence: A. Rory McQuiston, Department of Anatomy and Neurobiology, Virginia Commonwealth University, Box 980709, Richmond, VA, 23298 USA amcquiston@vcu.edu Received: 13 January 2015 Accepted: 12 March 2015 Published: 13 April 2015 ORIGINAL RESEARCH published: 13 April 2015 doi: 10.3389/fncel.2015.00115 Breeding Strategies and Chat Immunoﬂuorescence Two different breeding strategies were developed for these studies. Studies examining nicotinic responses in interneuron subtypes utilized a triple cross consisting of Chat-Cre × VIP-Cre × YFP-reporter (CVY). Studies that utilized archaerhodopsin to silence specific interneuron subtypes in CA1 used a triple cross consisting of Chat-Cre × VIP-Cre × Arch-GFP (CVA) (see Bell et al., 2015 for more details). Animals Viptm1(cre)Zjh/J (VIP-Cre, JAX Stock No. 010908), B6;129P2- Pvalbtm1(cre)Arbr/J (PV-cre, Jax Stock No. 008069), B6;129S6- Chattm1(cre)Lowl/J (Chat-Cre, JAX Stock No. 006410), B6.Cg-Gt(ROSA)26Sortm3(CAG-EYFP)Hze/J (YFP, Jax Stock No. 007903), and B6;129S-Gt(ROSA)26Sortm35.1(CAG-aop3/GFP)Hze/J (Arch-GFP, Jax Stock No. 012735) mice (Hippenmeyer et al., 2005; Madisen et al., 2010; Rossi et al., 2011; Taniguchi et al., 2011) used in these studies were housed in an animal care facility approved by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). Animal experimental procedures followed a protocol approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University (AD20205). This protocol adhered to the ethical guidelines described in The Care and Use of Laboratory Animals 8th Edition (Garber et al., 2011). All efforts were made to minimize animal suffering and to reduce the number of animals used. g In the mammalian central nervous system, 11 nicotinic receptor subunits have been identified, 9 of which have been found in the hippocampus (Sudweeks and Yakel, 2000). A significant portion of the influence that nicotinic receptor activation has on hippocampal network function is likely due to the expression of nicotinic receptors on inhibitory interneurons. In particular, α7 (Alkondon et al., 1997; Jones and Yakel, 1997; Frazier et al., 1998b; McQuiston and Madison, 1999), α4β2∗(McQuiston and Madison, 1999; Sudweeks and Yakel, 2000) and α2 nicotinic receptors (McQuiston and Madison, 1999; Sudweeks and Yakel, 2000; Jia et al., 2009) have been shown to be functionally expressed on various subsets of hippocampal interneurons. Furthermore, electrically-evoked release of Acetylcholine (ACh) has been shown to result in α7 nicotinic receptor mediated excitatory postsynaptic responses in CA1 interneurons (Alkondon et al., 1998; Frazier et al., 1998a). Based on the identification of α7-mediated nicotinic synaptic events and the prevalence of functional α7 nicotinic responses in interneurons, α7 subunits had been thought to be the primary subunits mediating nicotinic receptor activation of hippocampal inhibitory interneurons. However, more recent studies that utilized optogenetics to release ACh from medial septum/diagonal band of Broca (MS/DBB) axon terminals in mouse hippocampal CA1 showed that nicotinic EPSPs in interneurons were almost exclusively mediated by α4β2∗ nicotinic receptors (Bell et al., 2011). These latter studies fit with behavioral studies that suggested that α4β2∗nicotinic receptors may have a significant role in nicotine enhancement of memory processing (Davis and Gould, 2006). Introduction In terms of aging and neurodegenerative disease, a significant loss of α4 nicotinic receptor subunit expression has been observed in aging mice (Gahring et al., 2005) and an 80% decrease in α4 subunit expression has been reported in Alzheimer’s patients (Kellar et al., 1987). Furthermore, beta amyloid protein, a protein that has been associated with the etiology of Alzheimer’s disease, can inhibit α4β2∗receptors at low concentrations (Wu et al., 2004). Therefore, it appears that α4β2∗nicotinic receptor function in the hippocampus is necessary for normal memory formation and their dysfunction may contribute to memory impairment associated with aging and neurodegenerative disease. receptors on hippocampal CA1 interneurons can be activated by ACh release from MS/DBB terminals (Bell et al., 2011), the identity of the specific subsets of interneurons displaying α4β2∗ nicotinic responses remains unknown. Therefore, using Cre-driver mice, fluorescent protein reporter mice, and optogenetics, we have investigated the subsets of hippocampal CA1 interneurons that respond with nicotinic responses following the release of ACh. Introduction The formation of new memories in the hippocampus is influenced by nicotinic receptor function. This is exemplified by observations that the encoding of memories can be enhanced by the exogenous activation of nicotinic receptors (Davis and Gould, 2006; Levin et al., 2009) April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org Nicotinic transmission onto CA1 interneurons Bell et al. and memory performance can be impaired by the injection of α4β2∗(nicotinic receptors that contain α4 and β2 subunits but may include other types of subunits) or α7 nicotinic receptor antagonists directly into the hippocampus (Levin, 2002). Furthermore, dysfunction of both α4 and β2 nicotinic subunits in the hippocampus have been correlated with memory impairment associated with addiction, neurodegenerative disease and aging. In regard to nicotine addiction, the β2 nicotinic subunit in the hippocampus has been linked to chronic nicotine withdrawal (Davis and Gould, 2009) and high affinity nicotinic receptors in the hippocampus, which are likely composed of α4β2∗subunits (Nguyen et al., 2004), are upregulated in smokers (Perry et al., 1999). In terms of aging and neurodegenerative disease, a significant loss of α4 nicotinic receptor subunit expression has been observed in aging mice (Gahring et al., 2005) and an 80% decrease in α4 subunit expression has been reported in Alzheimer’s patients (Kellar et al., 1987). Furthermore, beta amyloid protein, a protein that has been associated with the etiology of Alzheimer’s disease, can inhibit α4β2∗receptors at low concentrations (Wu et al., 2004). Therefore, it appears that α4β2∗nicotinic receptor function in the hippocampus is necessary for normal memory formation and their dysfunction may contribute to memory impairment associated with aging and neurodegenerative disease. and memory performance can be impaired by the injection of α4β2∗(nicotinic receptors that contain α4 and β2 subunits but may include other types of subunits) or α7 nicotinic receptor antagonists directly into the hippocampus (Levin, 2002). Furthermore, dysfunction of both α4 and β2 nicotinic subunits in the hippocampus have been correlated with memory impairment associated with addiction, neurodegenerative disease and aging. In regard to nicotine addiction, the β2 nicotinic subunit in the hippocampus has been linked to chronic nicotine withdrawal (Davis and Gould, 2009) and high affinity nicotinic receptors in the hippocampus, which are likely composed of α4β2∗subunits (Nguyen et al., 2004), are upregulated in smokers (Perry et al., 1999). Frontiers in Cellular Neuroscience \| www.frontiersin.org Preparation of Hippocampal Slices Brain slices were obtained by methods previously described (Bell et al., 2011). In brief, horizontal slices containing the mid- temporal hippocampus were cut at 350–450 µm on a Leica VT1200 (Leica Microsystems, Buffalo Grove, IL). Sections were incubated in a holding chamber kept at 36◦C for 30 min and then allowed to return to room temperature. The holding and recording chamber solution consisted of normal saline (in mM): NaCl 125, KCl 3.0, CaCl2 1.2, MgSO4 1.2, NaHPO4 1.2, NaHCO3 25, glucose 25 bubbled with 95% O2/5% CO2. Recordings were performed at 32–35◦C. Ampliﬁcation of Fluorescent Markers Slices were fixed in 4% paraformaldehyde (Boston BioProducts) and incubated with streptavidin Alexa Fluor 633 (Life Technologies, Invitrogen) in phosphate buffered saline (PBS) with Triton-X 100 as previously described (Bell et al., 2011). Processed slices were then reconstructed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). Alexa Fluor 633 was excited with the 633 nm line of a HeNe 5 mW laser and cells were visualized using a 20× dry lens (0.8 N.A., voxel dimensions 0.2 × 0.2 × 1.1 µm). The imaged interneurons were traced using the Autoneuron module within the Neurolucida program (MBP, Burlington, VT). For amplification of YFP-labeled interneurons, 1:200 dilution of rabbit anti-GFP conjugated to Alexa Fluor 488 (Life Technologies, Invitrogen) in goat blocking buffer (10% normal serum, 2% bovine serum albumin, 0.4% Triton-X 100 in 0.1 M phosphate buffer) was added to fixed and washed slices for overnight incubation. Before and after primary and secondary antibody incubations, slices were washed in PBS. Slices were mounted in either Prolong Gold® (Life Technologies, Invitrogen) or VECTASHIELD® hard mount (Vector Laboratories). Generation and Stereotaxic Injection of rAAV-Flex-rev-oChIEF-tdTomato into the MS/DBB of Chat-Cre Mice In total, 7 × 100 nl injections were made between 3.75 and 5.0 mm in depth. For injections into the left nucleus basalis, coordinates were AP = −0.5, Lat = −1.6, Depth = 5.0–4.2 mm, and injection volume was 100 nl/site every 200 µm for a total of 500 nl. 10–15 days post viral injection, 42–70 day old mice were sacrificed for experimentation. Light-Evoked Release of ACh from MS/DBB Cholinergic Axon Terminals and Light-Evoked Silencing of CA1 Interneuron Subtype Populations p Cholinergic terminals expressing oChIEF-tdTomato were stimulated by blue light and interneurons expressing Arch- GFP were hyperpolarized by yellow light. Both light paths were transmitted through the epi-illumination light path of an Olympus BX51WI microscope and a 10× water immersion objective (0.3 NA). Blue light flashes (1 ms in duration) and yellow light pulses (4 s in duration) were generated from light-emitting diodes (LEDs) (UHP-microscope-LED-460 or UHP-T-LED-White filtered by an HQ 575/50x excitation filter, respectively, Prizmatix Modiin-Ilite, Givat Shmuel, Israel). Blue or yellow light exiting the LEDs were reflected or passed through a dichroic mirror (515dcxru, Chroma Technology, Bellows Falls, VT, USA) and were focused into the epi-illumination light path of the Olympus BX51WI microscope and back aperture of the 10x water immersion objective (0.3 NA) using an optiblock beam combiner (Prizmatix) and a dichroic mirror (700dcxxr, Chroma Technology, Bellows Falls, VT, USA) in the filter turret. Generation and Stereotaxic Injection of rAAV-Flex-rev-oChIEF-tdTomato into the MS/DBB of Chat-Cre Mice A recombinant adeno-associated virus (rAAV, serotype 1, 1.05 or 1.8 × 1013 VC/ml titer) expressing FLEXed oChIEF-tdTomato was generated using a previously described method (Bell et al., 2011) in order to selectively express oChIEF-tdTomato in infected cells that also expressed Cre recombinase. Mice were initially anesthetized via intraperitoneal injection of ketamine (100 mg/kg IP) and xylazine (2.5 mg/kg IP). Anesthesia was maintained with O2 supplemented with isoflurane. For injections into the MS/DBB, an incision was made in the skin along the midsagittal suture, and a small hole was drilled in the skull overlying the septum. An aluminosilicate glass pipette containing rAAV-Flex-rev-oChIEF-tdTomato was lowered to the level of the MS/DBB, 1.0 mm rostral to Given the importance of α4β2∗nicotinic receptors in normal and pathophysiological nervous system function, it is crucial to identify neuronal types endogenously activated by nicotinic receptors. Although it is known that α4β2∗nicotinic April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 2 Nicotinic transmission onto CA1 interneurons Bell et al. in voltage clamped current recordings (Vh = −70 mV). Otherwise, a standard KGluc and lower BAPTAK4 intracellular solutions where used to measure membrane potential responses. Membrane potentials and/or currents were measured with a Model 2400 patch clamp amplifier (A-M Systems, Port Angeles, WA) and converted into a digital signal by a PCI-6040E A/D board (National instruments, Austin, TX). WCP Strathclyde Software was used to store and analyze membrane potential and current responses on a PC computer (courtesy of Dr. J Dempster, Strathclyde University, Glasgow, Scotland). To detect and analyze spontaneous inhibitory postsynaptic currents (sIPSCs), miniAnalysis (Synaptosoft, Fort Lee, NJ) was used. Further analysis was performed with Originpro 8.1 (OriginLab Corp., Northampton, MA, USA), Excel (Microsoft, Redmond, WA) and SPSS 20.0 (IBM, Armonk, NY). Bregma and infused at a rate of 100 nl/min using a software driven injectomate (Neurostar, Sindelfingen, Germany). In total, 7 × 100 nl injections were made between 3.75 and 5.0 mm in depth. For injections into the left nucleus basalis, coordinates were AP = −0.5, Lat = −1.6, Depth = 5.0–4.2 mm, and injection volume was 100 nl/site every 200 µm for a total of 500 nl. 10–15 days post viral injection, 42–70 day old mice were sacrificed for experimentation. Bregma and infused at a rate of 100 nl/min using a software driven injectomate (Neurostar, Sindelfingen, Germany). Electrophysiological Measurements Data were analyzed using WCP software and miniAnalysis for the electrophysiological measurements. Statistics were performed using SPSS 20.0 (IBM, Armonk, NY). Statistical significances for groups of 3 or more were determined using a one-way ANOVA with Bonferroni post hoc tests. The averaged statistical significances for groups of 2 were determined with two-tailed t-tests. For averaged time-dependent sIPSC frequency data, a one-way ANOVA was done to test whether the averaged sIPSC frequency changed over the course of each experiment. Differences were determined to be statistically significant for p values less than 0.05. All data was reported as the mean, standard error of the mean (SEM). Asterisks were p y g Whole cell patch clamp recordings from hippocampal CA1 interneurons were performed using patch pipettes (2–5 MΩ) pulled from borosilicate glass (8250 1.65/1.0 mm) on a Narishige PC-10 pipette puller filled with (in mM): KCl 55, KGluc 70, NaCl 8, MgATP 2, NaGTP 0.1, HEPES 10, BAPTAK4 2, QX314 chloride 10, biocytin 0.1% or KGluc 130, NaCl 8, MgATP 2, NaGTP 0.1, HEPES 10, BAPTAK4 0.1, biocytin 0.1%. Elevated intracellular KCl, BAPTAK4, and the inclusion of QX314 in the intracellular solution were used for the measurement of spontaneous inhibitory postsynaptic currents (IPSCs), which appear as inward or negative going currents April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 3 Nicotinic transmission onto CA1 interneurons Bell et al. as follows unless otherwise noted, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. as follows unless otherwise noted, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. optogenetically released ACh resulted in depolarizations that increased in amplitude in the presence of atropine (Figures 1C, 2A–E,I). The nicotinic responses were not significantly affected by bath application of the α7 nicotinic receptor antagonist MLA (25 nM, Figures 1A,B, gray, n = 12), but they were blocked by bath application of the α4β2∗nicotinic receptor antagonist DHβE (1 µM, Figures 1A,B, orange, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.001, n = 20) suggesting that they were mediated by α4β2∗nicotinic receptors (Bell et al., 2011). Anatomical reconstruction of some nicotinic responding interneurons displayed morphology similar to that described for type 2 VIP/IS interneurons (Figure 1A; Acsády et al., 1996a) whereas others could not be definitively correlated with previously described classes of VIP/IS interneurons because of limited axonal morphology. Electrophysiological properties of nicotinic responding VIP interneurons were diverse. Chemicals All chemicals were purchased from VWR unless otherwise indicated. VU 10010 (M4-selective positive allosteric modulator), SR 95531 hydrobromide (Gabazine, GABAA antagonist), Baclofen (GABAB antagonist), QX314 chloride (intracellular sodium channel blocker), and AF-DX 116 (selective M2- muscarinic receptor antagonist) were obtained from Tocris Bioscience (Ellisville, Missouri) and 6, 7-Dinitroquinoxaline-2, 3-dione (DNQX, AMPA receptor antagonist), DL-2-Amino-5- phosphono pentanoic acid (APV, NMDA receptor antagonist) from Ascent Scientific (Bristol, U.K.). Biocytin (B-1592) was purchased from Life Technologies (Invitrogen). Electrophysiological Measurements The average resting membrane potential for nicotinic responding VIP interneurons was −73.9 mV ± 0.8 mV (n = 30) and the average input resistance was 395.3 MΩ, ± 20.8 MΩ(n = 30). The nicotinic responsive interneurons showed no consistent active membrane properties. Some interneurons displayed irregular action potential firing patterns (Figures 1D–F) whereas others had regular accommodating action potential firing patterns (Figure 1G) (average accommodation ratio (last interval/first interval) 2.75 ± 0.84, n = 30). Many nicotinic receptor responding interneurons had decreasing action potential amplitudes during depolarizing test pulses (ratio last/first = 0.34, ± 0.07, n = 30) (Figures 1D,F). Furthermore, different nicotinic responding VIP interneurons could either display a voltage sag (Figure 1G) or it could be absent during hyperpolarizations (Figures 1D,F) (average sag ratio of 1.09 ± 0.01, n = 30). Therefore, nicotinic responding VIP interneurons could not be classified based on electrophysiological properties. In contrast to VIP/IS interneurons, endogenous nicotinic excitatory responses were not observed in VIP basket cells, parvalbumin interneurons, or pyramidal neurons (Bell et al., 2015, data not shown). Thus, nicotinic responses resulting from ACh release may preferentially occur in interneuron-selective interneurons. Frontiers in Cellular Neuroscience \| www.frontiersin.org Results ACh Released from MS/DBB Terminals Selectively Produced α4β2∗Nicotinic Responses in VIP Interneuron-Selective Interneurons There are two types interneurons that express vasoactive- intestinal peptide (VIP) in hippocampal CA1: those that exclusively innervate other interneurons (interneuron-selective interneurons, VIP/IS) and those that innervate the perisomatic region of pyramidal neurons (VIP basket cells, VIP/BC) (Acsády et al., 1996b). While nicotinic responses appear to occur in neocortical VIP interneurons (Arroyo et al., 2012), little is known about how hippocampal VIP interneurons respond to ACh released from MS/DBB cholinergic terminals. Therefore, we investigated the actions of ACh release on VIP interneurons using whole cell patch clamp recordings and optogenetics in acute mouse hippocampal brain slices. To target whole cell patch clamp recordings from VIP interneurons, we utilized CVY animals (see methods) that expressed YFP in VIP interneurons. To optogenetically release ACh from MS/DBB cholinergic terminals in hippocampal brain slices, we expressed the excitatory optogenetic protein oChIEF-tdTomato in MS/DBB cholinergic neurons through Cre-dependent AAV mediated transfection. Following whole cell patch clamp measurements, interneurons were morphologically reconstructed to determine if the interneuron from which we recorded was either a perisomatic projecting basket cell or an interneuron-selective interneuron (Figure 1A, VIP/IS). Cells with incomplete morphology were labeled ‘‘noID’’ (VIP/noID). Nicotinic Responses in VIP/IS Interneurons are Presynaptically Inhibited by M2 Autoreceptors Presynaptically Inhibited by M2 Autoreceptors In order determine whether muscarinic receptors contributed to depolarizing responses in VIP/IS interneurons, we bath applied atropine to inhibit muscarinic receptors. Rather than inhibiting the depolarization, atropine potentiated this response (Bell et al., 2011). This potentiation was independent of the number (10—Figure 2A vs. 120—Figures 2B,C) or frequency of pulses (20 Hz—Figures 2A,B vs. 8 Hz—Figure 2C). In some VIP/IS interneurons (5 of 20), previously subthreshold nicotinic responses could become suprathreshold following the application of atropine (Figures 2A–C). On average, the presence of atropine resulted in a 283 ± 30% increase in the area of the response (Figure 2D, t-test, p < 0.001, n = 20). To test whether this potentiation was due to a presynaptic or postsynaptic mechanism, we blocked postsynaptic G-protein coupled signaling by substituting GTP with GDP-β-S in the In a previous study, we demonstrated that all morphologically identified VIP basket cells responded to ACh release with a muscarinic receptor-dependent slow depolarization completely inhibited by atropine (Bell et al., 2015). In the present study, we attempted to preferentially record from VIP/IS cells by targeting recordings to small VIP fluorescent neurons in the middle of the stratum lacunosum-moleculare (SLM). These SLM interneurons had smaller cell bodies (≤10 um) that have been correlated with VIP/IS cells and not VIP basket cells (Acsády et al., 1996a). In most VIP/IS cells (19 of 21) and all small cell bodied SLM VIP/noID cells (15 of 15) April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 4 Nicotinic transmission onto CA1 interneurons Bell et al. Bell et al. FIGURE 1 \| Acetylcholine (ACh) release activates α4β2∗-nicotinic receptors on vasoactive-intestinal peptide (VIP) interneuron-selective interneurons. (A) VIP interneuron-selective (IS) interneuron (morphology, A1) responded to optogenetically released ACh with fast depolarizations (black traces, 10 × 20 Hz) that were inhibited by 1 µM DHβE (orange traces) but not 25 nM MLA (gray trace). (B) All VIP interneurons not displaying basket cell morphology were unaffected by bath application of 25 nM MLA (gray bar, n = 12) but were blocked by 1 µM DHβE (orange bar, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.001, n = 20). (C) Histogram illustrating the distribution of response types across VIP expressing interneurons: VIP/IS cells, and VIP/noID. Most VIP/IS cells (19 of 21) and all non-visually identiﬁed VIP cells (19 of 19) exhibited nicotinic-dependent depolarizations (purple). Nicotinic Responses in VIP/IS Interneurons are Presynaptically Inhibited by M2 Autoreceptors Most VIP/IS cells (19 of 21) and all non-visually identiﬁed VIP cells (19 of 19) exhibited nicotinic-dependent depolarizations (purple). (D–F) Example of VIP interneurons with nicotinic responses that displayed irregular action potential ﬁring patterns that decreased in amplitude in response to depolarizing current injection. Negative current pulses produced hyperpolarizing membrane responses with little or no voltage sag. (G) A VIP interneuron that produced accommodating regular action potential ﬁring patterns to depolarizing current injection and a hyperpolarizing sag in response to negative current injection. interneurons. (A) VIP interneuron-selective (IS) interneuron (morphology, A1) responded to optogenetically released ACh with fast depolarizations (black traces, 10 × 20 Hz) that were inhibited by 1 µM DHβE (orange traces) but not 25 nM MLA (gray trace). (B) All VIP interneurons not displaying basket cell morphology were unaffected by bath application of 25 nM MLA (gray bar, n = 12) but were blocked by 1 µM DHβE (orange bar, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.001, n = 20). (C) Histogram illustrating the intracellular solution. When postsynaptic G-protein signaling was blocked, atropine continued to potentiate the nicotinic response (Figures 2E,I, one-way ANOVA, p < 0.001, Bonferroni post hoc test p < 0.001, n = 7) suggesting that ACh release from MS/DBB terminals onto nicotinic receptors is inhibited by presynaptic muscarinic autoreceptors. VU 10010 affected the nicotinic response. M4 receptors appeared to have no role in mediating presynaptic inhibition as VU 10010 (5 µM) had no effect on nicotinic response amplitudes (Figures 2G,I one-way ANOVA, ns, n = 5). In contrast, AF- DX 116 (500 nM) (Figures 2F,I) significantly increased the size of nicotinic responses suggesting that ACh release from MS/DBB terminals onto VIP/IS interneurons was inhibited by presynaptic M2 receptors (Figure 2I, one-way ANOVA, p < 0.001, Bonferroni post hoc test p < 0.001, n = 6). The magnitude of the potentiation by AF-DX116 was similar to that produced by atropine (Figure 2I) suggesting M2 receptors were involved. Furthermore, prior application of AF- DX116 occluded the effect of atropine on nicotinic response amplitudes (Figures 2H,I, t-test, ns, n = 6). Therefore, M2 muscarinic receptors mediate most if not all of the inhibition of ACh release. We next determined whether GDP-β-S effectively blocked postsynaptic G-protein coupled signaling in our experimental system. To do this we bath applied the GABAB agonist baclofen (10 µM) and measured hyperpolarizing responses in VIP/IS interneurons. Nicotinic Responses in VIP/IS Interneurons are Presynaptically Inhibited by M2 Autoreceptors In all cells where GTP was included in the intracellular solution (Figures 2J,K, black), baclofen caused a large sustained hyperpolarization (average amplitude at 4.5 min application = −11.6 ± 0.8 mV, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.01 for time points 4–7 min, n = 4). In contrast, baclofen had no significant effect on the membrane potential of VIP/IS interneurons in which GDP-β-S was included in the intracellular solution (average amplitude at 4.5 min application = −1.1 ± 0.9 mV, Figures 2J,K, blue, one-way ANOVA, ns, n = 4). Therefore, inclusion of GDP-β-S in the intracellular solution was effective in blocking postsynaptic G-protein coupled signaling and the atropine mediated potentiation of the nicotinic response was at least in part mediated by a presynaptic mechanism. Frontiers in Cellular Neuroscience \| www.frontiersin.org Nicotinic Responses in VIP/IS Interneurons are Presynaptically Inhibited by M2 Autoreceptors (D–F) Example of VIP interneurons with nicotinic responses that displayed irregular action potential ﬁring patterns that decreased in amplitude in response to depolarizing current injection. Negative current pulses produced hyperpolarizing membrane responses with little or no voltage sag. (G) A VIP interneuron that produced accommodating regular action potential ﬁring patterns to depolarizing current injection and a hyperpolarizing sag in response to negative current injection. FIGURE 1 \| Acetylcholine (ACh) release activates α4β2∗-nicotinic receptors on vasoactive-intestinal peptide (VIP) interneuron-selective interneurons. (A) VIP interneuron-selective (IS) interneuron (morphology, A1) responded to optogenetically released ACh with fast depolarizations (black traces, 10 × 20 Hz) that were inhibited by 1 µM DHβE (orange traces) but not 25 nM MLA (gray trace). (B) All VIP interneurons not displaying basket cell morphology were unaffected by bath application of 25 nM MLA (gray bar, n = 12) but were blocked by 1 µM DHβE (orange bar, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.001, n = 20). (C) Histogram illustrating the FIGURE 1 \| Acetylcholine (ACh) release activates α4β2∗-nicotinic receptors on vasoactive-intestinal peptide (VIP) interneuron-selective distribution of response types across VIP expressing interneurons: VIP/IS cells, and VIP/noID. Most VIP/IS cells (19 of 21) and all non-visually identiﬁed VIP cells (19 of 19) exhibited nicotinic-dependent depolarizations (purple). (D–F) Example of VIP interneurons with nicotinic responses that displayed irregular action potential ﬁring patterns that decreased in amplitude in response to depolarizing current injection. Negative current pulses produced hyperpolarizing membrane responses with little or no voltage sag. (G) A VIP interneuron that produced accommodating regular action potential ﬁring patterns to depolarizing current injection and a hyperpolarizing sag in response to negative current injection. distribution of response types across VIP expressing interneurons: VIP/IS cells, and VIP/noID. Most VIP/IS cells (19 of 21) and all non-visually identiﬁed VIP cells (19 of 19) exhibited nicotinic-dependent depolarizations (purple). (D–F) Example of VIP interneurons with nicotinic responses that displayed irregular action potential ﬁring patterns that decreased in amplitude in response to depolarizing current injection. Negative current pulses produced hyperpolarizing membrane responses with little or no voltage sag. (G) A VIP interneuron that produced accommodating regular action potential ﬁring patterns to depolarizing current injection and a hyperpolarizing sag in response to negative current injection. distribution of response types across VIP expressing interneurons: VIP/IS cells, and VIP/noID. Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation (A–C) Optogenetically released ACh produced depolarizing responses to short/fast (A, 10 × 20 Hz), prolonged/fast (B, 120 × 20 Hz), and prolonged/slow (C, 120 × 8 Hz) blue light ﬂashes (black traces). The depolarizations were potentiated by 10 µM atropine (green traces). (D) Atropine (green) signiﬁcantly potentiated the area of the nicotinic response (normalized to control, black, 10 × 20 Hz) (t-test, p < 0.001, n = 0). (E). Inclusion of GDP-β-S (black trace) in the intracellular recording solution did not inhibit atropine (green trace) potentiation of the nicotinic responses. (F) M2 antagonist AF-DX 116 (500 nM, red trace) potentiated nicotinic responses when GDP-β-S was included in the intracellular solution. (G) M4 positive allosteric modulator VU 10010 did not affect nicotinic responses (one-way ANOVA, ns, n = 5). (H) M2 antagonist AF-DX 116 (500 nM, red trace) occluded atropine (green trace) potentiation of nicotinic responses. (I) Histogram showing that atropine (GDP-β-S) (one-way ANOVA, p < 0.001, Bonferroni post hoc test p < 0.001, n = 7) and AF-DX 116 (GDP-β-S) (Bonferroni post hoc test p < 0.001, n = 6) signiﬁcantly potentiated nicotinic responses. Atropine (green checkers) did not signiﬁcantly increase nicotinic responses previously potentiated by AF-DX 116 (GDP-β-S) (t-test, ns, n = 6). (J) Application of 10 µM baclofen hyperpolarized VIP interneurons (black trace) but not when GDP-β-S (blue trace) was included in the intracellular solution. (K) All VIP interneurons tested produced signiﬁcant hyperpolarizations when exposed to baclofen (black dots, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.01 for time points 4–7 min, n = 4). Recordings that included GDP-β-S in the patch pipette did not respond to baclofen application (blue dots, one-way ANOVA, ns, n = 4). light flashes (Figure 3D, one-way ANOVA, p < 0.01, n = 23). This increase in frequency was subsequently blocked by bath application of DHβE (1 µM) suggesting that α4β2∗nicotinic receptors mediated the excitation of presynaptic interneurons (Figure 3D, one-way ANOVA, ns relative to control, n = 8). In order to determine whether VIP interneurons were the exclusive presynaptic interneurons activated by ACh release, Arch-GFP was expressed in VIP interneurons (Bell et al., 2015). When Arch was expressed in VIP-Cre interneurons, we found that a yellow light pulse did not affect the increased frequency of sIPSCs produced by ACh release (Figures 3F–H, one-way ANOVA, ns, n = 21). Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation We next investigated the downstream effects of the interneurons that were excited by nicotinic receptor activation. We recorded changes produced by nicotinic receptor activation on the frequency of sIPSCs in different CA1 interneuron subtypes located in the dendritic layers of CA1 (Figures 3A–H), CA1 pyramidal cells (Figures 3I–L), and layer 2/3 neocortical pyramidal neurons (Figures 3M–P). To increase the amplitudes of sIPSCs and improve their detection, recordings were made To examine which muscarinic receptor subtype mediated this presynaptic inhibition, we tested whether the M2-selective antagonist AF-DX 116 or the M4 positive allosteric modulator April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 5 Nicotinic transmission onto CA1 interneurons Bell et al. Bell et al. FIGURE 2 \| Presynaptic M2 autoreceptors inhibit the release of ACh onto α4β2∗nicotinic receptors in VIP/IS interneurons. (A–C) Optogenetically released ACh produced depolarizing responses to short/fast (A, 10 × 20 Hz), prolonged/fast (B, 120 × 20 Hz), and prolonged/slow (C, 120 × 8 Hz) blue light ﬂashes (black traces). The depolarizations were potentiated by 10 µM atropine (green traces). (D) Atropine (green) signiﬁcantly potentiated the area of the nicotinic response (normalized to control, black, 10 × 20 Hz) (t-test, p < 0.001, n = 0). (E). Inclusion of GDP-β-S (black trace) in the intracellular recording solution did not inhibit atropine (green trace) potentiation of the nicotinic responses. (F) M2 antagonist AF-DX 116 (500 nM, red trace) potentiated nicotinic responses when GDP-β-S was included in the intracellular solution. (G) M4 positive allosteric modulator VU 10010 did not affect nicotinic responses (one-way ANOVA, ns, n = 5). (H) M2 antagonist AF-DX 116 (500 nM, red trace) occluded atropine (green trace) potentiation of nicotinic responses. (I) Histogram showing that atropine (GDP-β-S) (one-way ANOVA, p < 0.001, Bonferroni post hoc test p < 0.001, n = 7) and AF-DX 116 (GDP-β-S) (Bonferroni post hoc test p < 0.001, n = 6) signiﬁcantly potentiated nicotinic responses. Atropine (green checkers) did not signiﬁcantly increase nicotinic responses previously potentiated by AF-DX 116 (GDP-β-S) (t-test, ns, n = 6). (J) Application of 10 µM baclofen hyperpolarized VIP interneurons (black trace) but not when GDP-β-S (blue trace) was included in the intracellular solution. (K) All VIP interneurons tested produced signiﬁcant hyperpolarizations when exposed to baclofen (black dots, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.01 for time points 4–7 min, n = 4). Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation Recordings that included GDP-β-S in the patch pipette did not respond to baclofen application (blue dots, one-way ANOVA, ns, n = 4). antagonist AF-DX 116 (500 nM, red trace) occluded atropine (green trace) potentiation of nicotinic responses. (I) Histogram showing that atropine antagonist AF-DX 116 (500 nM, red trace) occluded atropine (green trace) potentiation of nicotinic responses. (I) Histogram showing that atropine (GDP-β-S) (one-way ANOVA, p < 0.001, Bonferroni post hoc test p < 0.001, n = 7) and AF-DX 116 (GDP-β-S) (Bonferroni post hoc test p < 0.001, n = 6) signiﬁcantly potentiated nicotinic responses. Atropine (green checkers) did not signiﬁcantly increase nicotinic responses previously potentiated by AF-DX 116 (GDP-β-S) (t-test, ns, n = 6). (J) Application of 10 µM baclofen hyperpolarized VIP interneurons (black trace) but not when GDP-β-S (blue trace) was included in the intracellular solution. (K) All VIP interneurons tested produced signiﬁcant hyperpolarizations when exposed to baclofen (black dots, one-way ANOVA, p < 0.001, Bonferroni post hoc p < 0.01 for time points 4–7 min, n = 4). Recordings that included GDP-β-S in the patch pipette did not respond to baclofen application (blue dots, one-way ANOVA, ns, n = 4). FIGURE 2 \| Presynaptic M2 autoreceptors inhibit the release of ACh onto α4β2∗nicotinic receptors in VIP/IS interneurons. FIGURE 2 \| Presynaptic M2 autoreceptors inhibit the release of ACh onto α4β2∗nicotinic receptors in VIP/IS interneurons. (A–C) Optogenetically released ACh produced depolarizing responses to short/fast (A, 10 × 20 Hz), prolonged/fast (B, 120 × 20 Hz), and prolonged/slow (C, 120 × 8 Hz) blue light ﬂashes (black traces). The depolarizations were potentiated by 10 µM atropine (green traces). (D) Atropine (green) signiﬁcantly potentiated the area of the nicotinic response (normalized to control, black, 10 × 20 Hz) (t-test, p < 0.001, n = 0). (E). Inclusion of GDP-β-S (black trace) in the intracellular recording solution did not inhibit atropine (green trace) potentiation of the nicotinic responses. (F) M2 antagonist AF-DX 116 (500 nM, red trace) potentiated nicotinic responses when GDP-β-S was included in the intracellular solution. (G) M4 positive allosteric modulator VU 10010 did not affect nicotinic responses (one-way ANOVA, ns, n = 5). (H) M2 FIGURE 2 \| Presynaptic M2 autoreceptors inhibit the release of ACh onto α4β2∗nicotinic receptors in VIP/IS interneurons. Frontiers in Cellular Neuroscience \| www.frontiersin.org Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation (N) Voltage clamp recordings from a neocortical pyramidal neuron demonstrated that ACh release produced an increase in sIPSC frequency. (O) PSTH demonstrated that the time-dependent frequency of sIPSCs in the neocortical pyramidal neuron increased following ACh release. (P) The averaged time-dependent sIPSC frequency in all measured neocortical pyramidal neurons increased following ACh release (black line, gray shading = S.E.M., one-way ANOVA, p < 0.001, n = 5) and was blocked by 1 µM DHβE (orange line, orange shading = S.E.M., one-way ANOVA, ns, n = 5). ACh release persisted in the presence of yellow light (yellow bar). (H) The increased averaged time-dependent sIPSC frequency measured across all non-VIP interneurons (black line, gray shading = S.E.M, one-way ANOVA, p < 0.001, n = 21) was not suppressed by activation of Arch in VIP interneurons. (J) Voltage clamp recordings from a CA1 pyramidal neuron showed no change in sIPSC frequency following ACh release (blue bars). (K) PSTH demonstrated that the time-dependent sIPSC frequency was unchanged following ACh release in an individual CA1 pyramidal neuron. (L) Averaged time-dependent sIPSC frequency across all CA1 pyramidal cell recordings demonstrated no change sIPSC frequency following ACh release (black line, gray shading = S.E.M, one-way ANOVA, ns, n = 42). (N) Voltage clamp recordings from a neocortical pyramidal neuron demonstrated that ACh release produced an increase in sIPSC frequency. (O) PSTH demonstrated that the time-dependent frequency of sIPSCs in the neocortical pyramidal neuron increased following ACh release. (P) The averaged time-dependent sIPSC frequency in all measured neocortical pyramidal neurons increased following ACh release (black line, gray shading = S.E.M., one-way ANOVA, p < 0.001, n = 5) and was blocked by 1 µM DHβE (orange line, orange shading = S.E.M., one-way ANOVA, ns, n = 5). FIGURE 3 \| ACh release drives nicotinic receptor-mediated feedforward inhibition onto CA1 interneurons but not CA1 pyramidal neurons. (A,E,I,M) Schematic drawings of four recording paradigms. sIPSCs were recorded in response to ACh release in non-VIP CA1 interneurons located at the border of SR and SLM (A–H), in CA1 pyramidal neurons (I–L), or in layer 2/3 neocortical pyramidal neurons (M–P). All recordings were performed in the presence of 10 µM atropine, 30 µM DNQX, and 50 µM APV. (B) Voltage clamp recordings (Vh = −70 mV) demonstrating that optogenetic release of ACh (blue bars, 10 × 20 Hz) increased the number of sIPSCs observed in CA1 non-VIP interneurons. Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation This suggested that other non-VIP/IS interneurons also respond to ACh release through the activation of α4β2∗nicotinic receptors. with elevated intracellular chloride, QX314 to block action potentials that escaped voltage clamp, and elevated BAPTA to maintain low intracellular calcium levels. Under these recording conditions, voltage clamped sIPSCs resulted in inward currents. In all experiments, muscarinic receptors were blocked by atropine, ionotropic glutamate receptors were blocked by DNQX (30 µM) and APV (50 µM), and GABAB receptors were blocked by CGP54626 (1 µM). ACh was released by 10 × 1 ms flashes of blue light at 20 Hz at 1 min intervals. Peristimulus-time histograms for sIPSCs were constructed to detect any changes in the average time-dependent sIPSC frequency. Following the activation of nicotinic receptors by the optogenetic release of ACh, approximately half of dendritically located hippocampal CA1 interneurons (23 of 49 non VIP expressing, morphologically unidentified) displayed an increase in average sIPSC frequency (Figures 3B–D). One example illustrated that ACh release caused the time-dependent sIPSC frequency to increase from 0.035–0.231 Hz (Figure 3C). When normalized and averaged across all interneuron recordings, the average time-dependent sIPSC frequency increased by 574% (± 283%) during and immediately after 500 (ms after last flash) Hippocampal CA1 interneurons are inhibited by interneuron-selective interneurons as well as interneurons that innervate both interneurons and pyramidal cells. Therefore, we examined whether the non-VIP interneurons activated by nicotinic receptors inhibited pyramidal neurons in addition to interneurons. In all morphologically identified CA1 pyramidal neurons examined (n = 42), no change in time-dependent sIPSC April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 6 Nicotinic transmission onto CA1 interneurons Bell et al. ACh release persisted in the presence of yellow light (yellow bar). (H) The increased averaged time-dependent sIPSC frequency measured across all non-VIP interneurons (black line, gray shading = S.E.M, one-way ANOVA, p < 0.001, n = 21) was not suppressed by activation of Arch in VIP interneurons. (J) Voltage clamp recordings from a CA1 pyramidal neuron showed no change in sIPSC frequency following ACh release (blue bars). (K) PSTH demonstrated that the time-dependent sIPSC frequency was unchanged following ACh release in an individual CA1 pyramidal neuron. (L) Averaged time-dependent sIPSC frequency across all CA1 pyramidal cell recordings demonstrated no change sIPSC frequency following ACh release (black line, gray shading = S.E.M, one-way ANOVA, ns, n = 42). Discussion Our data suggest that nicotinic transmission preferentially engages specific networks in hippocampal CA1. Without affecting pyramidal neurons, nicotinic cholinergic transmission activated very specific groups of interneurons in hippocampal CA1 that selectively inhibit other interneurons (interneuron- selective interneurons). Release of ACh appeared to activate nicotinic receptors on both VIP and non-VIP interneuron- selective interneurons. Importantly, nicotinic responses were potently controlled by M2 muscarinic receptor-driven presynaptic inhibition. p y p Our data confirmed previous reports from our laboratory that suggested that hippocampal CA1 interneurons can be excited by ACh released onto postsynaptic α4β2∗nicotinic receptors (Bell et al., 2011). Our current studies extend these findings by demonstrating that α4β2∗-mediated nicotinic responses preferentially occurred in morphologically identified VIP/IS interneurons. We did not observe nicotinic responses in VIP/BCs, PV interneurons or pyramidal neurons. Furthermore, disynaptic IPSCs produced by nicotinic receptor activation were only detected in interneurons and not pyramidal cells. Importantly, these disynaptic IPSCs were not inhibited by optogenetic silencing of VIP interneurons suggesting that other non-VIP-expressing interneuron-selective interneurons were also excited by the release of ACh onto α4β2∗nicotinic receptors (Acsády et al., 1996b; Gulyás et al., 1996). This may be expected as VIP/IS interneurons only make up a minority (37%) of interneuron-selective interneurons (Acsády et al., 1996a). Importantly, a significant portion of the interneurons innervated by interneuron-selective interneurons target the dendrites of CA1 pyramidal neurons (Acsády et al., 1996a; Gulyás et al., 1996). Therefore, nicotinic disinhibition in hippocampal CA1 may facilitate dendritic integration of synaptic inputs in CA1 pyramidal neurons. Nicotinic receptor-mediated transmission in hippocampal CA1 has been shown to be potently inhibited by the release of ACh onto muscarinic receptors (Bell et al., 2011). Here we demonstrated that the muscarinic receptor inhibition of nicotinic transmission persisted when postsynaptic G-protein functioning was blocked—suggesting a presynaptic mechanism. This presynaptic inhibition of ACh release appears to be mediated by M2 muscarinic receptors, similar to findings in the thalamus (Sun et al., 2013). Importantly, muscarinic receptor presynaptic inhibition was potent and often prevented nicotinic responses from eliciting action potentials in interneuron- selective interneurons. Thus, therapeutic pharmacological blockade of M2 muscarinic receptors would be expected to facilitate nicotinic receptor-mediated influence on disinhibitory circuitry in hippocampal CA1. Similar to our findings in the hippocampus, nicotinic receptor-mediated disinhibition has been observed in the auditory cortex (Letzkus et al., 2011). Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation (C) A peristimulus time histogram (PSTH) illustrating the increase in time-dependent sIPSC frequency in nonVIP interneurons following ACh release. (D) An increase in the averaged time-dependent sIPSC frequency across all non-VIP interneurons (black line, gray shading = S.E.M, one-way ANOVA compared to baseline, p < 0.01, n = 23) was completely blocked by 1 µM DHβE (orange line, orange shading = SEM, one-way ANOVA, ns compared to baseline, n = 8). (F) Voltage clamp recordings showing that suppression of VIP interneurons by yellow light activation of Arch (yellow bar) did not inhibit the increase in sIPSC frequency following ACh release (blue bars). (G) PSTH showing time-dependent sIPSC frequency following FIGURE 3 \| ACh release drives nicotinic receptor-mediated feedforward inhibition onto CA1 interneurons but not CA1 pyramidal somewhat unexpected as previous studies had suggested that ACh release may activate nicotinic receptors on interneurons that innervate CA1 pyramidal neurons (Nagode et al., 2011). Furthermore, neocortical interneurons that innervate layer 2/3 pyramidal neurons have been shown to be excited by ACh release onto nicotinic receptors (Arroyo et al., 2012). frequency was observed during or following the release of ACh (Figures 3J–L). Furthermore, normalization and averaging the time-dependent sIPSC frequency across all pyramidal neurons showed no significant increase in sIPSC frequency following ACh release compared to baseline sIPSC frequency (Figure 3L, one-way ANOVA, ns, n = 42). These observations were April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 7 Nicotinic transmission onto CA1 interneurons Bell et al. Therefore, to test whether in our system we can activate nicotinic receptors on interneurons that innervate pyramidal neurons, we expressed oChIEF-tdTomato in cholinergic neurons of the nucleus basalis. This permitted us to record from layer 2/3 pyramidal neurons of the neocortex and measure changes in sIPSC frequency following ACh release (Figures 3N–P). Similar to previous reports (Arroyo et al., 2012), ACh release increased the time-dependent frequency of sIPSC in neocortical pyramidal neurons (Figures 3N–P, one-way ANOVA, p < 0.001, n = 5) and the increased frequency was blocked by DHβE (1 µM, one-way ANOVA, ns relative to control, n = 5). Therefore, our data suggested that ACh release onto nicotinic receptors in hippocampal CA1 preferentially activates interneurons that innervate other interneurons rather than interneurons that inhibit pyramidal neurons. of cholinergic terminals. However, activation of Arch in our preparation had no effect on the nicotinic driven inhibitory postsynaptic currents measured in interneurons. Postsynaptic Targets of Interneurons Excited by Nicotinic Receptor Activation Thus, Arch must not be expressed at sufficient concentrations in cholinergic terminals to influence optogenetically-driven release of ACh. p g y Our nicotinic data were inconsistent with previous findings that suggested that CA1 pyramidal neurons (∼20%) (Gu and Yakel, 2011) and interneurons that innervate pyramidal neurons (Nagode et al., 2011) may be excited by ACh released onto nicotinic receptors. Although we have never observed a nicotinic response in recordings from 132 pyramidal neurons or 104 non IS interneuron subtypes, it is possible that we did not sample enough neurons or had a bias toward recording from particular interneuron subtypes. Furthermore, different injection methods and excitatory optogenetic proteins used between the different studies may have limited our ability to record nicotinic responses in pyramidal neurons or interneurons that innervated pyramidal cells. Alternatively, the studies that reported nicotinic responses in other cell types did not confirm the recorded neurons identity with post hoc anatomical reconstructions or electrophysiological characterization. Therefore, it remains possible that the nicotinic responses (Gu and Yakel, 2011) and nicotinic receptor-driven disynaptic IPSCs (Nagode et al., 2011) measured in putative pyramidal neurons were actually recordings made from interneurons and not pyramidal cells. Another possibility for the lack of nicotinic receptor-driven disynaptic IPSCs in pyramidal neurons observed in our study may be that nicotinic responses in interneurons that target pyramidal neurons are mostly subthreshold. Regardless of these inconsistencies, our data suggest that a primary effect of α4β2∗nicotinic receptor-mediated transmission is to excite interneuron-selective interneurons and aid in disinhibition of the hippocampal CA1 network. Frontiers in Cellular Neuroscience \| www.frontiersin.org References Acsády, L., Arabadzisz, D., and Freund, T. F. (1996a). Correlated morphological and neurochemical features identify different subsets of vasoactive intestinal polypeptide-immunoreactive interneurons in rat hippocampus. Neuroscience 73, 299–315. doi: 10.1016/0306-4522(95)00610-9 Frazier, C. J., Rollins, Y. D., Breese, C. R., Leonard, S., Freedman, R., and Dunwiddie, T. V. (1998b). Acetylcholine activates an alpha-bungarotoxin- sensitive nicotinic current in rat hippocampal interneurons, but not pyramidal cells. J. Neurosci. 18, 1187–1195. Acsády, L., Görcs, T. J., and Freund, T. F. (1996b). Different populations of vasoactive intestinal polypeptide-immunoreactive interneurons are specialized to control pyramidal cells or interneurons in the hippocampus. Neuroscience 73, 317–334. doi: 10.1016/0306-4522(95) 00609-5 Gahring, L. C., Persiyanov, K., and Rogers, S. W. (2005). Mouse strain-specific changes in nicotinic receptor expression with age. Neurobiol. Aging 26, 973–980. doi: 10.1016/j.neurobiolaging.2004.07.005 Garber, J. C., Barbee, R. W., Bielitzki, J. T., Clayton, L. A., Donovan, J. C., Kohn, D. F., et al. (2011). Guide for the Care and Use of Laboratory Animals. 8th Edn. Washington, D.C.: National Academies Press. Alkondon, M., Pereira, E. F., and Albuquerque, E. X. (1998). α-bungarotoxin- and methyllycaconitine-sensitive nicotinic receptors mediate fast synaptic transmission in interneurons of rat hippocampal slices. Brain Res. 810, 257–263. doi: 10.1016/s0006-8993(98)00880-4 Gonchar, Y., Wang, Q., and Burkhalter, A. (2008). Multiple distinct subtypes of GABAergic neurons in mouse visual cortex identified by triple immunostaining. Front. Neuroanat. 1:3. doi: 10.3389/neuro.05.003.2007 Alkondon, M., Pereira, E. F., Barbosa, C. T., and Albuquerque, E. X. (1997). Neuronal nicotinic acetylcholine receptor activation modulates gamma- aminobutyric acid release from CA1 neurons of rat hippocampal slices. J. Pharmacol. Exp. Ther. 283, 1396–1411. Gu, Z., and Yakel, J. L. (2011). Timing-dependent septal cholinergic induction of dynamic hippocampal synaptic plasticity. Neuron 71, 155–165. doi: 10.1016/j. neuron.2011.04.026 Arroyo, S., Bennett, C., Aziz, D., Brown, S. P., and Hestrin, S. (2012). Prolonged disynaptic inhibition in the cortex mediated by slow, non-alpha7 nicotinic excitation of a specific subset of cortical interneurons. J. Neurosci. 32, 3859–3864. doi: 10.1523/JNEUROSCI.0115-12.2012 Gulyás, A. I., Hájos, N., and Freund, T. F. (1996). Interneurons containing calretinin are specialized to control other interneurons in the rat hippocampus. J. Neurosci. 16, 3397–3411. Hippenmeyer, S., Vrieseling, E., Sigrist, M., Portmann, T., Laengle, C., Ladle, D. R., et al. (2005). A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol. 3:e159. doi: 10.1371/journal. pbio.0030159 Bell, L. A., Bell, K. A., and McQuiston, A. R. (2015). Summary Optogenetic techniques have recently permitted more extensive studies on the effect of ACh release in various regions of the CNS. In particular, nicotinic responses to ACh release have been studied in several brain regions including the interpeduncular nucleus (Ren et al., 2011), hippocampus (Bell et al., 2011), striatum (English et al., 2011), neocortex (Letzkus et al., 2011; Arroyo et al., 2012), spinal cord (Lamotte d’Incamps et al., 2012) and thalamus (Sun et al., 2013). A common feature of these responses is that they activate GABAergic neurons. In some areas, nicotinic receptors activate GABAergic projection neurons (Ren et al., 2011; Sun et al., 2013). In other regions, they activate select types of inhibitory interneurons (Bell et al., 2011; English et al., 2011; Arroyo et al., 2012). The Acknowledgments The authors would like to thank Drs. John Lin and Roger Tsien for donating oChIEF-tdTomato cDNA and Scott Sternson for rAAV-FLEX-rev-ChR2-tdTomato. We would also like to thank Dr. John Dempster for the gift of his Strathclyde Electrophysiological Software. These studies were supported by grants from the National Institutes of Health (1R01MH094626-01 and 1R21MH103695-01). The authors declare no competing financial interests. Frazier, C. J., Buhler, A. V., Weiner, J. L., and Dunwiddie, T. V. (1998a). Synaptic potentials mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal interneurons. J. Neurosci. 18, 8228–8235. Discussion Some of the neocortical interneurons excited by ACh release expressed Chat, which has been shown to be expressed exclusively in VIP-expressing interneurons (Porter et al., 1998; Gonchar et al., 2008). However, unlike our studies, nicotinic transmission was observed in interneurons that innervated both pyramidal neurons and other interneurons (Arroyo et al., 2012). Therefore, the effect of It should be noted that the triple crosses in the experiments described above should result in the expression of Arch-GFP in both VIP and cholinergic neurons (CVA animals). This could make interpretation of the results from these experiments difficult as an inhibition of the nicotinic receptor-driven response might result from the inhibition of VIP neurons or the inhibition April 2015 \| Volume 9 \| Article 115 8 Nicotinic transmission onto CA1 interneurons Bell et al. nicotinic receptor-mediated transmission in the neocortex will likely be more complex than a primarily disinhibitory role that we hypothesize for the hippocampus. impact that physiologically activated nicotinic receptors have in each brain region will depend on the specific types of GABAergic neurons activated by ACh release. In the case of hippocampal CA1, interneuron-selective interneurons appear to be preferentially activated by nicotinic receptors suggesting a feedforward disinhibitory role for nicotinic receptor-mediated transmission. References Nicotinic α7- or β2-containing receptor knockout: effects on radial-arm maze learning and long-term nicotine consumption in mice. Behav. 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Taniguchi, H., He, M., Wu, P., Kim, S., Paik, R., Sugino, K., et al. (2011). A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013. doi: 10.1016/j.neuron.2011. 07.026 Nagode, D. A., Tang, A. H., Karson, M. A., Klugmann, M., and Alger, B. E. (2011). Optogenetic release of ACh induces rhythmic bursts of perisomatic IPSCs in hippocampus. PLoS One 6:e27691. doi: 10.1371/journal.pone.0027691 Nguyen, H. N., Rasmussen, B. A., and Perry, D. C. (2004). Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment. J. Neurochem. 90, 40–49. doi: 10.1111/j.1471-4159.2004.02482.x Wu, J., Kuo, Y. P., George, A. A., Xu, L., Hu, J., and Lukas, R. J. (2004). β-amyloid directly inhibits human α4β2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells. J. Biol. 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(2009). α2 nicotine receptors function as a molecular switch to continuously excite a subset of interneurons in rat hippocampal circuits. Eur. J. Neurosci. 29, 1588–1603. doi: 10.1111/j. 1460-9568.2009.06706.x Bell, K. A., Shim, H., Chen, C. K., and McQuiston, A. R. (2011). Nicotinic excitatory postsynaptic potentials in hippocampal CA1 interneurons are predominantly mediated by nicotinic receptors that contain alpha4 and beta2 subunits. Neuropharmacology 61, 1379–1388. doi: 10.1016/j.neuropharm.2011. 08.024 Jones, S., and Yakel, J. L. (1997). Functional nicotinic ACh receptors on interneurones in the rat hippocampus. J. Physiol. 504(Pt. 3), 603–610. doi: 10. 1111/j.1469-7793.1997.603bd.x Davis, J. A., and Gould, T. J. (2006). The effects of DHBE and MLA on nicotine-induced enhancement of contextual fear conditioning in C57BL/6 mice. Psychopharmacology (Berl) 184, 345–352. doi: 10.1007/s00213- 005-0047-y Kellar, K. J., Whitehouse, P. J., Martino-Barrows, A. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Ren, J., Qin, C., Hu, F., Tan, J., Qiu, L., Zhao, S., et al. (2011). Habenula ‘‘cholinergic’’ neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes. Neuron 69, 445–452. doi: 10.1016/j.neuron.2010.12.038 April 2015 \| Volume 9 \| Article 115 Frontiers in Cellular Neuroscience \| www.frontiersin.org 10
https://openalex.org/W3113055872	https://www.researchsquare.com/article/rs-73053/v2.pdf?c=1631873146000	English	null	Takotsubo syndrome induced by brachytherapy in a patient with endocervical adenocarcinoma	Cardio-oncology	2,020	cc-by	3,256	Takotsubo Syndrome induced by brachytherapy in a patient with endocervical adenocarcinoma. Aline Cristini Vieira (  aline.cristini@hotmail.com ) Aline Cristini Vieira (  aline.cristini@hotmail.com ) Hospital Sirio-Libanes Short communication Posted Date: October 26th, 2020 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 on December 5th, 2020. See the published version at https://doi.org/10.1186/s40959-020-00082-8. Page 1/10 Abstract Background: Takotsubo syndrome (TTS), also known as stress cardiomyopathy, apical ballooning syndrome and broken heart syndrome, is characterized by acute-onset chest pain, electrocardiographic (ECG) abnormalities and reversible left ventricular (LV) disfunction in the absence of a culprit obstructive lesion in the coronary arteries; therefore, myocardial infarction is the most important differential diagnosis. Usually induced by emotional/physical stress, its treatment consists in hemodynamic support until complete and spontaneous recovery occurs, which is generally achieved within a few days to weeks. Cervical malignancies are an important public health issue in low/middle-income countries and, in the setting of locally advanced disease, concurrent chemoradiation followed by brachytherapy is considered the standard treatment, harboring curative potential. Background: Takotsubo syndrome (TTS), also known as st syndrome and broken heart syndrome, is characterized by a (ECG) abnormalities and reversible left ventricular (LV) disf lesion in the coronary arteries; therefore, myocardial infarct diagnosis. Usually induced by emotional/physical stress, it until complete and spontaneous recovery occurs, which is g Cervical malignancies are an important public health issue setting of locally advanced disease, concurrent chemoradia the standard treatment, harboring curative potential. Case report: We report a case of a 38-year-old woman who underwent concurrent chemoradiotherapy and developed cardiopulmonary arrest in ventricular fibrillation during a brachytherapy session. Complementary tests disclosed altered ECG and cardiac biomarkers, no evidence of coronary artery obstruction, as well as LV disfunction consistent with TTS on echocardiogram and cardiac MRI. After few days of supportive therapy, complete recovery of heart function was observed. Conclusion: Especially for cancer patients, who usually experience intense emotional/physical stress intrinsically associated with their diagnosis and aggressive treatments, considering TTS as a differential diagnosis is warranted. Intracavitary brachytherapy procedure may represent a trigger for TTS. Case Report A 38-year-old female presented with an abnormal pap smear in January 2018 without local or systemic manifestations. A biopsy was performed and the patient was diagnosed with endocervical adenocarcinoma. A staging magnetic resonance imaging (MRI) of the pelvis and a positron emission tomography – computed tomography (PET-CT) were performed, which showed a hypermetabolic bulky lesion arising from uterine cervix (SUV max: 23.0), without parametrial, bladder, rectal involvement, or distant metastasis. Clinical staging based on International Federation of Gynecology and Obstetrics (FIGO) classified the disease in FIGO IIA1. The patient started chemoradiotherapy with cisplatin 40mg/m2 weekly, and high dose-rate brachytherapy starting in the fourth week of external beam irradiation. At the end of the third session (four brachytherapy sessions programmed), during applicator removal, although under anesthesia, the patient developed a ventricular tachycardia with pulse, followed by cardiopulmonary arrest in ventricular fibrillation. After two minutes of cardiopulmonary resuscitation, return of spontaneous circulation was achieved, and the patient was admitted to an intensive care unit. During the investigation, ECG showed ST-segment elevation in lead II and in AVF, and prolonged QT interval of 470 msec (Figure 1). There was a rise in the levels of serum creatine kinase-MB (CKMB) level to 10.4 ng/mL (normal limit: < 5.0 ng/mL), serum troponin-I level was 2.04 ng/ml (normal limit: < 0.16ng/ml) and natriuretic peptide B levels (BNP) to 233 pg/mL (normal limit: < 100 pg/mL). Transthoracic echocardiogram showed marked LV dysfunction, with akinesia of all apical and middle segments of LV wall, with estimated LV ejection fraction (LVEF) of 30% (Figure.2). The coronary angiography demonstrated no obstructive lesions in the coronary arteries and moderate hypokinesia of the anterior and inferior apical segments of LV wall (Figure 3.). Holter showed rare and isolated polymorphic ventricular premature beats and signs of heart dysautonomia with parasympathetic depression and adrenergic predominance. A cardiac MRI was performed seven days after the event showing circumferential mid-ventricle hypokinesia associated with LV systolic dysfunction, however with a significant improvement of the global LV contractility (Figure 4A and Figure 4B), absence of myocardial fibrosis in the late gadolinium enhancement sequences (Figure 4C), signs of myocardial edema in the LV wall in T2-weighted sequences and moderate pericardial effusion (Figure 4D), suggestive with Takotsubo cardiomyopathy. Treatment with carvedilol 3.125 mg twice daily and enalapril 2.5 mg twice daily (titrating the dose up to the maximum dose tolerated by the patient) was initiated. Introduction Takotsubo syndrome (TTS), also known as stress cardiomyopathy, apical ballooning syndrome and broken heart syndrome, is an acute and transient left ventricular (LV) myocardial dysfunction, which can occur in the setting of a severe psychological or physical stress event, most often occurring 1 to 5 days before.1 TTS’s clinical presentation might be indistinguishable from an acute coronary syndrome (ACS) with respect to symptoms, electrocardiographic (ECG) changes and biomarkers. Since its first report in 1990 by Sato et al,2 TTS remains with no reliable non-invasive diagnostic approach, leaving coronary angiography with left ventriculography as the gold standard diagnostic tool to reject or to ratify this diagnosis to this days.3,4 Typically, it occurs in postmenopausal women with few cardiovascular risk factors.1,3–5 The exact pathophysiology of TTS is still unknown.6 Besides anecdotal case reports describing the occurrence of TTS in the setting of malignancy and chemotherapy, the role of chemotherapy and tumor in the development of TTS with regard to physical and emotional stress remains unclear.7 It has been suggested that the treatment of the malignancy itself is associated with the development of TTS.8 Herein we describe a case of a 38-year-old female patient without history of cardiovascular disorder who developed TTS during a brachytherapy session to treat an endocervical adenocarcinoma. Page 2/10 Case Report The levels of troponin-I returned to the normal range after 4 days, CKMB levels after 6 days and BNP in 12 days. Fifteen days after the event, the patient presented with chest pain, and pericarditis was diagnosed. Repeat echocardiogram after 12 days of the TTS event, showed remarkable improvement in LV function, with LVEF of 68%, preserved biventricular function and small pericardial effusion. The patient was treated with Ibuprofen 800 mg three times per day and Colchicine 0.5 mg twice daily for three months, with good response and no signs of recurrence. The cardiac MRI was repeated two months later, showing a preserved biventricular contractility, a Page 3/10 Page 3/10 Page 3/10 physiological pericardial effusion, and no late gadolinium enhancement suggestive of myocardial fibrosis. Once complete cardiac recovery was achieved, she was able to resume brachytherapy, and the remaining application was performed 15 days later as an inpatient condition and under rigorous monitoring, uneventfully. The total cancer treatment time was 64 days. physiological pericardial effusion, and no late gadolinium enhancement suggestive of myocardial fibrosis. Once complete cardiac recovery was achieved, she was able to resume brachytherapy, and the remaining application was performed 15 days later as an inpatient condition and under rigorous monitoring, uneventfully. The total cancer treatment time was 64 days. The patient received further adjuvant treatment with cisplatin 50 mg/m2 plus gemcitabine 100 mg/m2, with discontinuation after one cycle due to severe myelotoxicity. Further, she continued her therapy with carvedilol and enalapril for approximately 6 months when she spontaneously decided to interrupt the treatment. The patient remains in follow-up with sustained complete response from the cancer as demonstrated by pelvic MRI and clinical examination performed every six months for two years until the time that this manuscript was submitted to publication. Conclusion TTS appears to occur more frequently than was previously thought. Increased awareness of the existence of this syndrome and knowledge of its risk factors are gaining importance in recent years. Further research is required to investigate the interplay of malignancies, its treatments and TTS, which might be helpful to elucidate the pathophysiological mechanism of this cardiomyopathy. To the best of our knowledge, we are herein reporting the second case of TTS arising in the setting of radiation therapy and the first for brachytherapy. Discussion Endothelial dysfunction in epicardial and microvascular coronary arteries occurs frequently in patients with cancer, especially during and after systemic chemotherapy or radiotherapy of the heart region, which might be a predisposing factor for the developement of TTS.1,3,14 Mediastinal radiotherapy can induce heart disease, such as coronary obstruction, stenosis or regurgitation due to valvular fibrosis, cardiomyopathy and pericardial constriction and inflammation.15 Though cisplatin remains the cornerstone of endocervical carcinoma stage IIA1 treatment chemotherapy and a standard of care, however it is also related to some other rare side effects like vasculitis, cardiomyopathy and vascular thrombosis. Cisplatin inducing cardiomyopathy and thromboembolic phenomena can be explained by several mechanisms, some of which are endovascular injury with intimal fibrosis, decreased activation of protein C, hypomagnesaemia and myocardial fiber apoptosis11. Previous usage of cisplatin might have contributed for the development of myocardial dysfunction in our patient. Although, so far there is limited data on TTS induced by radiotherapy and no reports on brachytherapy- induced cardiomyopathy. In fact, brachytherapy by itself is painless. However, applicators placement and removal may present only mild discomfort or severe pain according to the different procedures (molds, intracavitary procedures, interstitial implants), and the use of sedation or anesthesia is properly indicated. TTS in the context of intracavitary gynecological brachytherapy procedure, not the radiation delivery, could have been triggered by the unweighted pain experienced by our patient during applicator removal. This case may represent a warning for intracavitary gynecological brachytherapy procedures, since in many facilities in our country it is performed only with sedation, or even without any sedation or analgesia. Although most patients with TTS experience complete cardiac function recovery, the complication rates can be compared to those found in patients with acute coronary syndrome.13 Patients who survive an acute episode typically recover systolic function within one to four weeks.1,3 Our patient recovered the systolic function within two weeks. Discussion This report illustrates the case of a young patient with locally advanced endocervical adenocarcinoma, FIGO stage IIA1, that was recommended to treatment with concurrent chemoradiotherapy and presented TTS followed by cardiorespiratory arrest induced during brachytherapy. TTS is clinically an acute myocardial infarction-like cardiomyopathy without a culprit coronary artery lesion.3 Approximately 80–90% of cases occur in postmenopausal women and classically TTS has been linked to emotional stress due to excessive release of catecholamines as mechanism of response.1,9,10 Although the patient in this case outwardly displayed very little anxiety about her diagnosis and treatment approach, it is possible that she felt a heightened degree of internal emotional stress that may have contributed to the development of TTS. It is also possible that she presented some degree of pain during applicator removal at the end of the third brachytherapy session, due to anesthesia superficialization, which could have triggered a cardiomyopathy induced by the release of several inflammatory cytokines and metabolites related to this physical distress. Of note, a small vaginal laceration was observed after the applicator removal. An important subject of our case is the differential diagnosis with myocarditis. The presence of pericardial effusion is not part of the diagnosis of TTS and it is very common in patients with myocarditis. The absence of late gadolinium enhancement by cardiac RMI may also be present in patients with myocarditis, especially in the initial days. However, the cardiac MRI performed 2 months later to the event did not demonstrate any sign of myocardial fibrosis, which helped to rule out the hypothesis of myocarditis. The relationship between malignancy and TTS is particularly interesting from an epidemiologic, mechanistic and outcome standpoint as both malignancy and chemotherapy have been associated with TTS.11 The overall long-term mortality of TTS patients with malignant disease is significantly increased and the prevalence of cancer in patients with TTS is high, considerably exceeding that in the normal population.12 One of the hypothesis is that cancer and TTS share similar triggering mechanisms, which Page 4/10 Page 4/10 consist in activation of the sympathetic nervous system.8,13 Therefore, TTS patients with cancer should be considered a high-risk subgroup with respect to their increased mortality rate. Declarations Page 5/10 Page 5/10 Page 5/10 Affiliations: 1. Oncology Center, Hospital Sírio Libanês, São Paulo, Brazil 2. Cardiology Center, Hospital Sírio-Libanês, São Paulo, Brazil 3. Department of Radiation Oncology, Hospital Sírio-Libanês, Brazil. 3. Department of Radiation Oncology, Hospital Sírio-Libanês, Brazil. 4. Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. Acknowledgements: Not applicable. Acknowledgements: Not applicable. Availability of data and materials: Not applicable. Availability of data and materials: Not applicable. Competing interests: The authors declare that they have no potential conflicts of interest related to this publication. Funding: None. Funding: None. Author’s Contributions: All authors contributed significantly to the manuscript. All authors provided critique and feedback on the manuscript. All authors read and approved the final version of the manuscript. Authors' information: Affiliations: Ethics approval and consent to participate: This case report was approved by the ethics committee of the Hospital Sírio-Libanês, São Paulo, Brazil. Consent for publication: The patient provided consent and gave permission to have her case, as well as relevant related workup and diagnostic images, presented in the medical literature. LVEF: Left ventricular ejection fraction LVEF: Left ventricular ejection fraction References 1. Medina de Chazal H, Del Buono MG, Keyser-Marcus L, et al. Stress Cardiomyopathy Diagnosis and Treatment. Journal of the American College of Cardiology. 2018;72(16):1955-1971. doi:10.1016/j.jacc.2018.07.072 2. Sato H. Tako-tsubo-like left ventricular dysfunction due to multivessel coronary spasm. In: Clinical Aspect of Myocardial Injury: From Ischemia to Heart Failure. Kagakuhyoronsha Publishing Co; 1990:56-64. 3. Ghadri J-R, Wittstein IS, Prasad A, et al. International Expert Consensus Document on Takotsubo Syndrome (Part I): Clinical Characteristics, Diagnostic Criteria, and Pathophysiology. European Heart Journal. 2018;39(22):2032-2046. doi:10.1093/eurheartj/ehy076 3. Ghadri J-R, Wittstein IS, Prasad A, et al. International Expert Consensus Document on Takotsubo Syndrome (Part I): Clinical Characteristics, Diagnostic Criteria, and Pathophysiology. European Heart Journal. 2018;39(22):2032-2046. doi:10.1093/eurheartj/ehy076 4. Ghadri J-R, Wittstein IS, Prasad A, et al. International Expert Consensus Document on Takotsubo Syndrome (Part II): Diagnostic Workup, Outcome, and Management. European Heart Journal. 2018;39(22):2047-2062. doi:10.1093/eurheartj/ehy077 4. Ghadri J-R, Wittstein IS, Prasad A, et al. International Expert Consensus Document on Takotsubo Syndrome (Part II): Diagnostic Workup, Outcome, and Management. European Heart Journal. 2018;39(22):2047-2062. doi:10.1093/eurheartj/ehy077 5. Ueyama T, Kasamatsu K, Hano T, Tsuruo Y, Ishikura F. Catecholamines and Estrogen Are Involved in the Pathogenesis of Emotional Stress-induced Acute Heart Attack. Annals of the New York Academy of Sciences. 2008;1148(1):479-485. doi:10.1196/annals.1410.079 5. Ueyama T, Kasamatsu K, Hano T, Tsuruo Y, Ishikura F. Catecholamines and Estrogen Are Involved in the Pathogenesis of Emotional Stress-induced Acute Heart Attack. Annals of the New York Academy of Sciences. 2008;1148(1):479-485. doi:10.1196/annals.1410.079 6. Ghadri JR, Ruschitzka F, Lüscher TF, Templin C. Takotsubo cardiomyopathy: still much more to learn. Heart. 2014;100(22):1804-1812. doi:10.1136/heartjnl-2013-304691 6. Ghadri JR, Ruschitzka F, Lüscher TF, Templin C. Takotsubo cardiomyopathy: still much more to learn. Heart. 2014;100(22):1804-1812. doi:10.1136/heartjnl-2013-304691 7. Toyooka S, Akagi S, Furukawa M, et al. Takotsubo cardiomyopathy associated with pulmonary resections after induction chemoradiotherapy for non-small cell lung cancer. Gen Thorac Cardiovasc Surg. 2012;60(9):599-602. doi:10.1007/s11748-012-0058-7 7. Toyooka S, Akagi S, Furukawa M, et al. Takotsubo cardiomyopathy associated with pulmonary resections after induction chemoradiotherapy for non-small cell lung cancer. Gen Thorac Cardiovasc Surg. 2012;60(9):599-602. doi:10.1007/s11748-012-0058-7 8. Munoz E, Iliescu G, Vejpongsa P, et al. Takotsubo Stress Cardiomyopathy. Journal of the American College of Cardiology. 2016;68(10):1143-1144. doi:10.1016/j.jacc.2016.06.027 8. Munoz E, Iliescu G, Vejpongsa P, et al. Takotsubo Stress Cardiomyopathy. Journal of the American College of Cardiology. 2016;68(10):1143-1144. doi:10.1016/j.jacc.2016.06.027 9. Ghadri JR, Kato K, Cammann VL, et al. Long-Term Prognosis of Patients With Takotsubo Syndrome. Journal of the American College of Cardiology. 2018;72(8):874-882. Abbreviations TTS: Takotsubo syndrome Page 6/10 MRI: Magnetic resonance imaging References doi:10.1016/j.jacc.2018.06.016 9. Ghadri JR, Kato K, Cammann VL, et al. Long-Term Prognosis of Patients With Takotsubo Syndrome. Journal of the American College of Cardiology. 2018;72(8):874-882. doi:10.1016/j.jacc.2018.06.016 10. Sharkey SW, Windenburg DC, Lesser JR, et al. Natural History and Expansive Clinical Profile of Stress (Tako-Tsubo) Cardiomyopathy. Journal of the American College of Cardiology. 2010;55(4):333-341. doi:10.1016/j.jacc.2009.08.057 11. Demkow U, Stelmaszczyk-Emmel A. Cardiotoxicity of cisplatin-based chemotherapy in advanced non-small cell lung cancer patients. Respiratory Physiology & Neurobiology. 2013;187(1):64-67. doi:10.1016/j.resp.2013.03.013 Page 7/10 Figures Figures Figure 4 Cine cardiac magnetic resonance images showing a 3-chamber view of the left ventricle in end diastole (A) and in end systole (B) demonstrating a circumferential mid-ventricle hypokinesia with greater intensity in the anteroseptal segment (arrows). C image shows late gadolinium enhancement sequence showing a 4-chamber view of the heart without any sign of myocardial fibrosis. (D) Cine cardiac magnetic resonance sequence demonstrating diffuse pericardium effusion of moderate intensity (arrows). (E) Short-tau inversion-recovery (STIR) sequence, a T2-weighted sequence used to assess myocardial edema, demonstrating the presence of hypersignal in the middle segments of the anterior, septal and lateral walls (arrows) which indicates the presence of myocardial edema in these segments. Archivewithcorrections.docx Figure 1 ECG tracing of the patient (25 mm/s, 10 mm/mV) showing ST-segment elevation on D2 and prolonged QT interval prolonged (QTc* 470 ms). QTc: corrected QT interval ECG tracing of the patient (25 mm/s, 10 mm/mV) showing ST-segment elevation on D2 and prolonged QT interval prolonged (QTc 470 ms). *QTc: corrected QT interval Figure 2 Apical 4 chambers view showing akinesia of the mid-apical segments of the inferior septal and anterolateral walls, determining apical ballooning (arrows). LA: Left Atrial. LV: Left Ventricle. RA: Right Atrial. RV: Right Ventricle. Figure 2 Apical 4 chambers view showing akinesia of the mid-apical segments of the inferior septal and anterolateral walls, determining apical ballooning (arrows). LA: Left Atrial. LV: Left Ventricle. RA: Right Atrial. RV: Right Ventricle. Apical 4 chambers view showing akinesia of the mid-apical segments of the inferior septal and anterolateral walls, determining apical ballooning (arrows). LA: Left Atrial. LV: Left Ventricle. RA: Right Atrial. RV: Right Ventricle. Page 8/10 Page 8/10 Figure 3 Cineangiocoronariography showing absent of significant obstructive atherosclerotic lesions. Left coronary in right anterior cranial oblique incidence (A). Right coronary in left anterior cranial oblique incidence (B). Cardiac ventriculography demonstrating mid left ventricular segments hypokinesia (arrows). Left ventricle in max diastole (C) and in max systole (D) in right oblique incidence. Figure 3 Cineangiocoronariography showing absent of significant obstructive atherosclerotic lesions. Left coronary in right anterior cranial oblique incidence (A). Right coronary in left anterior cranial oblique incidence (B). Cardiac ventriculography demonstrating mid left ventricular segments hypokinesia (arrows). Left ventricle in max diastole (C) and in max systole (D) in right oblique incidence. Cineangiocoronariography showing absent of significant obstructive atherosclerotic lesions. Left coronary in right anterior cranial oblique incidence (A). Right coronary in left anterior cranial oblique incidence (B). Cardiac ventriculography demonstrating mid left ventricular segments hypokinesia (arrows). Left ventricle in max diastole (C) and in max systole (D) in right oblique incidence. Page 9/10 Page 9/10 Figure 4 Cine cardiac magnetic resonance images showing a 3-chamber view of the left ventricle in end diastole (A) and in end systole (B) demonstrating a circumferential mid-ventricle hypokinesia with greater intensity in the anteroseptal segment (arrows). C image shows late gadolinium enhancement sequence showing a 4-chamber view of the heart without any sign of myocardial fibrosis. (D) Cine cardiac magnetic resonance sequence demonstrating diffuse pericardium effusion of moderate intensity (arrows). Figure 1 (E) Short-tau inversion-recovery (STIR) sequence, a T2-weighted sequence used to assess myocardial edema demonstrating the presence of hypersignal in the middle segments of the anterior, septal and lateral walls (arrows) which indicates the presence of myocardial edema in these segments. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 10/10
https://openalex.org/W3042733884	https://link.springer.com/content/pdf/10.1007/s00217-020-03564-2.pdf	English	null	Effect of sweeteners and storage on compositional and sensory properties of blackberry jams	European food research & technology	2,020	cc-by	12,969	Abstract Sweeteners of natural sources, such as sugar alcohols, are in the centre of growing interest. Their impact on the phytochemi- cals, antioxidant and sensory properties of blackberry jams were investigated during a 9-months storage period. Measure- ments on jams prepared with different sugars and sugar alcohols (sucrose, fructose, xylitol and erythritol) were performed at the date of preparation and in the 1st, 3rd, 6th, 9th month. Total polyphenol content, individual polyphenols, antioxidant properties, anthocyanin content and CIE Lab* colour were determined. Sensory profiles were determined by a trained panel. Sensory attributes were compared by the results of the electronic tongue and nose.if Sweeteners had a significant impact on physicochemical properties and sensory attributes. Storage time also affected the sensory and compositional properties of jams. Changes in antioxidant properties did not follow a clear trend during the storage period, and antioxidant capacity was not affected significantly by the sweetening agent, but showed a significant decline from the 6th month. A positive effect of xylitol was observed in terms of a low degradation rate of anthocyanins, while their decomposition was the fastest in the fructose-containing preparation. Jams sweetened with erythritol reached significantly lower values for some sensory attributes (blackberry flavour and general taste intensity), however, they showed more intense red colour. Multiple factor analysis enabled the identification of the effect of sweetener and storage time on the pattern of the sensory data matrix. Classification according to individual sweeteners was performed successfully by the electronic tongue, but not by electronic nose. Keywords Blackberry jam · Sweetener · Sugar alcohol · Polyphenol · Sensory analysis · Multiple factor analysis Keywords Blackberry jam · Sweetener · Sugar alcohol · Polyphenol · Sensory analysis · Multiple fac * Csilla Benedek benedek.csilla@se‑etk.hu 1 Faculty of Health Sciences, Department of Dietetics and Nutrition, Semmelweis University, Vas u. 17, 1088 Budapest, Hungary 2 Faculty of Food Science, Department of Physics and Control, Szent István University, Budapest, Hungary 3 Faculty of Health Sciences, Department of Dietetics and Nutrition, Semmelweis University, Budapest, Hungary 4 Faculty of Food Science, Department of Postharvest and Sensory Evaluation, Szent István University, Budapest, Hungary 5 Faculty of Food Science, Department of Refrigeration and Livestock Products’ Technology, Szent István University, Budapest, Hungary 6 Faculty of Food Science, Department of Applied Chemistry, Szent István University, Budapest, Hungary European Food Research and Technology (2020) 246:2187–2204 https://doi.org/10.1007/s00217-020-03564-2 European Food Research and Technology (2020) 246:2187–2204 https://doi.org/10.1007/s00217-020-03564-2 ORIGINAL PAPER ORIGINAL PAPER Csilla Benedek1 · Zsanett Bodor2 · Vanda Tímea Merrill3 · Zoltán Kókai4 · Attila Gere4 · Zoltan Kovacs2 · István Dalmadi5 · László Abrankó6 Csilla Benedek1 · Zsanett Bodor2 · Vanda Tímea Merrill3 · Zoltán Kókai4 · Attila Gere4 · Zoltan István Dalmadi5 · László Abrankó6 Received: 28 February 2020 / Revised: 2 July 2020 / Accepted: 4 July 2020 / Published online: 19 July 2020 © The Author(s) 2020 Introduction Sugar substitutes, especially natural sweeteners are getting more popular, in accordance with the trends of modern nutri- tion. Products with reduced energy or sugar content, as well as sugar-free products made with sugar alcohols are very successful on the market, which can be partly explained by the increasing consumer aversion towards artificial additives, including intensive sweeteners. They are also used on a large scale by the food industry due to their favourable sensory attributes and good technological properties as thickeners or humectants; they are resistant to heat and pH changes and are not involved in Maillard reaction. Although sugar alcohols are not a health concern for healthy individuals, showing some potential health benefits [1], undesired effects as bloating and diarrhoea may appear at some people at the consumption of high doses, especially those suffering of 1 Faculty of Health Sciences, Department of Dietetics and Nutrition, Semmelweis University, Vas u. 17, 1088 Budapest, Hungary 2 Faculty of Food Science, Department of Physics and Control, Szent István University, Budapest, Hungary 3 Faculty of Health Sciences, Department of Dietetics and Nutrition, Semmelweis University, Budapest, Hungary 4 Faculty of Food Science, Department of Postharvest and Sensory Evaluation, Szent István University, Budapest, Hungary 5 Faculty of Food Science, Department of Refrigeration and Livestock Products’ Technology, Szent István University, Budapest, Hungary 6 Faculty of Food Science, Department of Applied Chemistry, Szent István University, Budapest, Hungary (0123 1 3456789) 3 European Food Research and Technology (2020) 246:2187–2204 2188 bowel problems. Nevertheless, xylitol and erythritol are reported to be more tolerable than sorbitol or mannitol. Safety of polyols is currently being re-evaluated by the Euro- pean Food Safety Authority, this re-evaluation is planned to be completed by the end of 2020 [2]. As a sweetener alternative, fructose is still markedly present on the mar- ket of diabetic products as a low-cost sweetener with a low glycaemic index (GI). Its insulin-independent metabolism takes place directly in the liver, acting as a major mediator in the development of non-alcoholic fatty liver disease [3]. Due to the above-mentioned health-related limitations, the use of sugar alcohols, and especially fructose, requires due foresight. of pigments (anthocyanins in case of berries), interactions with other components of the matrix (including co-pigmen- tation), oxidative reactions; i.e., mainly Maillard process and caramelising, and oxidation of tannins [9]. Therefore, processing operations should be optimized not only for food safety but also for the nutritional aspects. Introduction Properly designed and monitored processing and storage of processed foods are decisive in increasing shelf-life without altering their nutritional value, colour attributes, and functionality [10]. It is also known that phenolic compounds contribute to the sensory attributes of fruits as well, thus their alteration during processing and storage will have an impact on many of the sensory characteristics of fruit preserves, including flavour, aroma and colour [7]. Nevertheless, only limited information is available on the effect of sugar alcohols on the preservation of these bioactive substances present in fruits and the physicochemical changes occurring during heat treatment and subsequent storage of the preserved products [12]. Berries and products containing berries, either in their fresh or preserved form, are popular amongst health-con- scious consumers due to their high levels of bioactive com- pounds, especially polyphenols. Polyphenol-rich diet has been associated with a range of health benefits, including anti-inflammatory effects, improvement of the endothelial and vascular function, improvement of fasting serum glu- cose, as well as beneficial effects on the gut microbiome and modulation of energy metabolism. Thus, polyphenols are effective in optimizing cardiometabolic health and reduce risk factors of type II diabetes and the metabolic syndrome and may protect against cognitive decline and dementia [4–7]. In the present research, the impact of some sugars (sucrose, as reference and fructose) and sugar alcohols (xylitol and erythritol) was studied on several characteris- tics of blackberry jams during storage, i.e., phytochemical (total polyphenol and anthocyanin) content, in vitro anti- oxidant capacity, colour and sensory properties. Objec- tives also included a comparison of sensory properties obtained by instrumental (electronic nose and tongue) and sensory profile analysis. There has been an increased concern over the past dec- ades regarding the protection of nutritional values and qual- ity optimization of processed foods, including the stabiliza- tion of polyphenols. At the same time, market trends clearly point towards preferences for low-calorie, sugar-free fruit preserves. Due to the favourable sensory and physiological properties of sugar alcohols, their combination with fruits is clearly present in the food industry. On the other hand, it is well known that the food matrix can also have a beneficial effect on berry anthocyanin stability [8]. However, the final colour of a food product containing anthocyanin-rich fruits is only partly determined by the initial anthocyanin content of the ingredient. Introduction Anthocyanin transformation and degrada- tion occurring during food processing operations and storage are complex processes that can significantly influence the actual colour of the product. Stability of anthocyanins is influenced by their skeleton, pH, light, temperature and the presence of complexing agents (proteins, metals) [9]. Thus, observed colour changes in processed foods are a composite result of various transformations of phenolic compounds in general, leading to yellowish or brownish pigments [10]. Darkening of the products is due to various oxidative reac- tions taking place during thermal treatment, such as oxida- tion of ascorbic acid or Maillard reaction, promoting dark pigments [11]. Thus, the overall colour alteration in ther- mally processed and stored products is probably due to a number of factors, such as degradation and polymerization Sensory profile analysis Sensory analyses were performed from freshly opened jars, at the date of preparation and at months 1, 3, 6, and 9, dis- tributing the samples from the same jar among panel mem- bers after homogenization. Samples were put in transparent glass bowls with 3-digit random sample codes. A simpli- fied profile analysis was applied during the sessions, i.e. the sensory descriptors (required by ISO 11,035) were previ- ously established (Fig. 1) and the 12 panellists were given a complete list of the attributes [20]. Panellists were trained according to ISO 8586:2012 in the Sensory Laboratory of Szent István University [21]. The sensory tests were carried out meeting all the criteria of the relevant ISO standards [22, 23]. Sample characteristics were compared to internal refer- ence values determined previously for the reference sample (sample containing sucrose). Neutral mineral water and neu- tral bakery products were used as taste neutralizers between evaluations. Values of sensory attributes were evaluated on a 0–100 unstructured line scale. At the end of the scoresheet, an open-ended textbox was provided to describe any addi- tional perceptions, attributes. Panellists worked in individual booths, with standard lighting, while data was captured with ProfiSens software [24]. Materials Fresh blackberry, sweeteners and pectin, as well as jars and caps were purchased from local supermarkets. All solvents and reagents were supplied by Sigma-Aldrich, except for potassium persulfate (Acros Organics), hydro- chloric acid (Carlo Erba) and distilled water. Crystalline polyphenols standards of caffeic acid, catechin, chloro- genic acid (5-caffeoyl-quinic acid, 5-CQA), epicatechin, ferulic acid, neochlorogenic acid (3-caffeoyl-quinic acid, 3-CQA), p-coumaric acid, quercetin-3-O-rutinoside (rutin) were purchased from Sigma-Aldrich/Merck (St. Louis, MI, USA), whereas cyanidin-glucoside (kuromanin), cyanidin- rutinoside (keracyanin), quercetin and quercetin-3-O-glu- coside from Extrasynthese (Genay, France). High purity water (Type1) was supplied by a Meck/Millipore Elix- Synergy water purification station. LC–MS grade formic acid and UHPLC-MS grade acetonitrile were purchased from VWR (Radnor, USA). 3 3 2189 European Food Research and Technology (2020) 246:2187–2204 points of each sample, obtaining thus five replicates. ΔE values were calculated according to the formula [19]: points of each sample, obtaining thus five replicates. ΔE values were calculated according to the formula [19]: Total soluble solids, total polyphenol, monomeric anthocyanin content and antioxidant properties of jams The percentage of soluble solids in terms of Brix was deter- mined according to the Annex of EU Regulation 974/2014/ EU, using a Kern Optics ORD 2UM digital refractometer [14]. Total polyphenol content (TPC, results expressed as gallic acid equivalents, GAE) [15] and total monomeric antho- cyanin content (pH differential method, results expressed as cyanidin-3-glucoside equivalents, using the molar absorptiv- ity value (ε) of 26,900 l/(molcm) [16] and ABTS + radical ion scavenging antioxidant capacity [17] (results expressed as trolox equivalents, TE) were determined spectrophoto- metrically on a Thermo Helios-α spectrophotometer, using proper dilutions (usually tenfold) of the sample extracts. Analyses were performed in five replicates per sample, using a sample extract prepared by adapting the method reported by Garcia-Viguera et al. (1997) [18]. Briefly, 2.00 g of jam was extracted for 30 min in an ultrasound bath using 10 ml of methanol:water:acetic acid 75:24.9:0.1. After centrifuga- tion (5 min, 4000 rpm), the supernatant was separated, and the extraction was repeated. The two supernatants were col- lected, adjusting the final volume to 20 ml with water. The average of the ten results (two extractions and five replicates and for each extract) was reported for each jam type (except for anthocyanin content, where three replicates were meas- ured per extract). Electronic tongue (ET) analysis Jam samples were analysed with the Alpha ASTREE poten- tiometric electronic tongue (ALPHA M.O.S., Toulouse, France). The sensor set composed of seven ISFET (ion- sensitive field-effect transistor) sensors (ZZ, CA, BB, GA, HA, JB, JE) that were designed to analyse and recognise the compounds in liquefied food [25]. The electronic tongue also contains a reference electrode (Ag/AgCl), and during the measurement the potential differences between the refer- ence and each working electrodes are detected. Results are derived from the average of the last 10 s of the individual sensors. Jam samples were measured in nine replicates. Jam preparation and storage Sweeteners, according to their sweetening power, were added to 1500 g of fresh fruit [13], i.e., 500 g sucrose (100%), 475 g fructose (105%), 500 g xylitol (100%) and 715 g erythritol (70%). 9 g pectin was also added to each preparation for consistency, then the mixtures were heated and boiled for 5 min. The jams resulted were distributed into previously sterilized jars, turned upside down for 10 min and then sterilized in a thermostat at 90 °C for 30 min and left there overnight. The jams were stored at room temperature in semi-dark conditions, modelling household conditions for 9 months, opening one bottle at the 0th, 1st, 3rd, 6th and 9th months for instrumental and sensory analyses. ΔE = √ (ΔL∗)2 + (Δa∗)2 + (Δb∗)2 ΔE = √ (ΔL∗)2 + (Δa∗)2 + (Δb∗)2 Colour determination Colorimetric measurements were determined in the CIE Lab tristimuli coordinate system with a Konica Minolta CR410 colorimeter. The instrument was standardized against a reference white plate (CR-A44) before sample measure- ments. Jam samples were spread out at 1 cm depth on a Petri plate, and the L, a, b* values were measured in at several Headspace analysis of samples was performed by an NST3320 type electronic nose (Applied Sensor, A.G., Sweden) with a built-in headspace autosampler unit for 12 samples. The sample chamber contains 23 different sen- sors, software for collecting and processing the data of the specimen. The NST 3320 is provided with 10 MOS-FET 1 3 3 European Food Research and Technology (2020) 246:2187–2204 2190 European Food Research and Technology (2020) 246:2187–2204 uctor field-effect transistor) sensors, emiconductor) sensors, and a sensor measurements. Ambient air was used e sensors, which was filtered through d a combined moisture/hydrocarbon started with sample equilibration at 30 °C for 15 min. Then reference air was pumped over the sensor surfaces for 10 s (baseline) followed by the infusion headspace for 20 s (sam- pling time) while the sensor signals were recorded. After sample analysis the recovery phase of the sensors was set e n- he riod 0 20 40 60 80 100 hue general odour intensity blackberry odour sweet odour sckiness general taste intensity blackberry ﬂavour sweet taste sour taste sucrose erythritol fructose xylitol 0 20 40 60 80 100 hue general odour intensity blackberry odour sweet odour sckiness general taste intensity blackberry ﬂavour sweet taste sour taste sucrose erythritol fructose xylitol a b Fig. 1 Sensory profile of the blackberry jams at the begin- ning (month 0) (a) and at the end (month 9) (b) of the period investigated 0 20 40 60 80 100 hue general odour intensity blackberry odour sweet odour sckiness general taste intensity blackberry ﬂavour sweet taste sour taste sucrose erythritol fructose xylitol a general odour intensity general taste intensity sckiness a 0 20 40 60 80 100 hue general odour intensity blackberry odour sweet odour sckiness general taste intensity blackberry ﬂavour sweet taste sour taste sucrose erythritol fructose xylitol b general odour intensity b (metal oxide semiconductor field-effect transistor) sensors, 12 MOS (metal oxide semiconductor) sensors, and a sensor for relative humidity measurements. Ambient air was used as a reference gas for the sensors, which was filtered through a silica gel column and a combined moisture/hydrocarbon filter. Fig. 1 Sensory profile of the blackberry jams at the begin- ning (month 0) (a) and at the end (month 9) (b) of the period investigated Identification of phenolic compounds by LC–MS Ultra-high performance liquid chromatography and electro- spray ionisation triple quadrupole tandem mass spectrom- etry (UHPLC-ESI–MS/MS) was used to monitor the fol- lowing 12 polyphenols: caffeic acid, catechin, chlorogenic acid (5-caffeoyl-quinic acid, 5-CQA), cyanidin-glucoside (kuromanin), cyanidin-rutinoside (keracyanin), epicat- echin, ferulic acid, neochlorogenic acid (3-caffeoyl-quinic acid, 3-CQA), p-coumaric acid, quercetin, quercetin-3-O- glucoside, quercetin-3-O-rutinoside (rutin). Quinic acid in 3-CQA and 5-CQA was considered as 1L-(–)-quinic acid 3R, 5R-(1α, 3α, 4α, 5β) [26] and its caffeoyl derivatives were assigned using the IUPAC 1976 numbering of cyclitols [27]. Compounds were separated on a Phenomenex Kinetex EVO C18 100 × 2.1 mm, 100A, 2.6 μm C18 column (plus similar pre-column) with gradient elution using high purity water containing 0.1% formic acid (mobile phase A) and UHPLC-MS grade acetonitrile (mobile phase B). Flow rate was 0.5 mL/min and the injection volume was 10 µL. Gradi- ent profile was as follows: 0 min 5% B; 4 min 60% B; 4.1 min 100% B; 5.1 min 100% B and 1 min post time. Column was kept at 30 °C. Analysis was carried out in 6.1 min on an Agi- lent Infinity II UHPLC coupled to an Ultivo ESI QQQ MS/ MS system. Mass spectrometric detection was performed in positive ion mode for kuromanin and keracyanin and in negative ion mode for the remaining ten compounds, apply- ing the dynamic multiple reaction (dMRM) scanning mode. Method was previously optimised with reference standards for each analyte. Matrix-matched calibration was used for quantification and sample extract (0.5 g–20 mL) was fivefold diluted in mobile phase ‘A’ and filtered through a 0.22 µm PTFE syringe filter before injection. Ultra-high performance liquid chromatography and electro- spray ionisation triple quadrupole tandem mass spectrom- etry (UHPLC-ESI–MS/MS) was used to monitor the fol- lowing 12 polyphenols: caffeic acid, catechin, chlorogenic acid (5-caffeoyl-quinic acid, 5-CQA), cyanidin-glucoside (kuromanin), cyanidin-rutinoside (keracyanin), epicat- echin, ferulic acid, neochlorogenic acid (3-caffeoyl-quinic acid, 3-CQA), p-coumaric acid, quercetin, quercetin-3-O- glucoside, quercetin-3-O-rutinoside (rutin). Quinic acid in 3-CQA and 5-CQA was considered as 1L-(–)-quinic acid 3R, 5R-(1α, 3α, 4α, 5β) [26] and its caffeoyl derivatives were assigned using the IUPAC 1976 numbering of cyclitols [27]. i In the case of the electronic tongue and nose results, outli- ers were identified based on the exploratory data evaluation (principal component analysis, PCA) and omitted before further data evaluation. Colour determination The gas flow rate of the dynamic sampling was set to 50 ml/min. The electronic nose measurement sequence started with sample equilibration at 30 °C for 15 min. Then reference air was pumped over the sensor surfaces for 10 s (baseline) followed by the infusion headspace for 20 s (sam- pling time) while the sensor signals were recorded. After sample analysis, the recovery phase of the sensors was set to 260 s including the flush time of the gas lines (60 s) with filtered air prior to the next sample injection to allow the 1 3 European Food Research and Technology (2020) 246:2187–2204 2191 of freedom available for the calculations was too low. The test also did not provide sufficient results for the monomeric anthocyanins and individual phenolic compounds due to the lower number of observations and thus the eligible degrees of freedom. However, the assumptions of the test were ana- lysed, i.e. elimination of outliers, normality and Mauchly’s sphericity test. In the case of significant Mauchly’s test of sphericity, the results were corrected using the Green- house–Geisser test (when p < 0.75). The pairwise compari- sons were done using the Bonferroni adjustment at p < 0.05 significance level [28]. re-establishment of the instrument baseline. The total cycle time per sample was 300 s. Each sample was measured 3 times and the results of nine measurements from three rep- licate samples were used for the statistical analysis. Identification of phenolic compounds by LC–MS As raw data pre-treatment, drift correction was applied in the case of ET to decrease the impact of the sensor drift during the different days [29]. Lin- ear discriminant analysis (LDA) was applied to build models for the classification of groups according to the sweeten- ing agents based on the results of ET and EN validated by three-fold cross-validation. Microsoft Excel 365 was used for the descriptive statistics, while R-project 3.5.2 software was used for processing the data of the EN and ET. Pro- file analysis results were evaluated using ProfiSens v. 2012 software [24]. The two-way repeated-measures ANOVA test was performed in IBM SPSS statistics software (Amonk, New York, USA). Sensory analysis data was evaluated using multiple factor analysis (MFA) to conduct a joint analysis of the five sensory sessions. MFA conducts individual PCAs on each of the data matrix and then merges them; hence enabling the researcher to analyse and visualize multiple data matrices as a whole. MFA was run using the XL-Stat software (Addinsoft, Long Island, NY, USA) [30]. Statistical evaluation Refractometry did not show a clear increment of the Brix values (Table 1), therefore, we could assume that there were no substantial losses due to evaporation, thus variations of the measured physicochemical and sensory parameters reflect the changes that occurred as a result of the chemical processes related to storage. At the first step, results were analysed using descriptive sta- tistics; average and standard deviation were calculated after outlier detection. Sensory data were screened for outliers using the z-score method. Outlier data was substituted with the group averages. Significant difference within different factors such as the storage time and sweetening agent were performed using two-way repeated-measures ANOVA. Due to the number of the observations this test was performed only in the case of the Lab, ΔE, total polyphenol content, ABTS antioxidant capacity and the results of the sensory profile analysis. It was not possible to evaluate the inter- action between the two factors as the number of degrees Global variation in total polyphenol content was fol- lowed by Folin–Ciocalteu assay during the whole storage period (Table 1). Statistical evaluation After an initial drop in the first 3 months (from 6.55 to 5.73 mg GAE/g for sucrose-, from 7.91 to 6.85 mg GAE/g for fructose-, from 7.50 to 5.80 mg GAE/g for xylitol- and from 6.85 to 6.13 mg GAE/g for erythri- tol-containing jams), an increase was observed for all the 1 3 European Food Research and Technology (2020) 246:2187–2204 2192 Table 1 Total polyphenol content, ABTS antioxidant capacity, total monomeric anthocyanin content and L, a, b and ΔE colour values of jams Mean ± standard deviation Sweet- ener/ month Total soluble solids (Brix, %) Total polyphe- nol content (mg GAE/g jam) ABTS antioxidant capacity (μmol TE / g jam) Total monomeric anthocyanins (mg cyanidin-3-gluco- side equivalents /100 g jam) L* a* b* ΔE Sucrose 0 36.25 ± 0.17 6.55 ± 0.35 82.59 ± 3.44 74.03 ± 3.56 29.21 ± 0.14 4.02 ± 0.05 − 0.46 ± 0.02 – 1 36.60 ± 0.12 6.09 ± 0.22 82.17 ± 2.88 59.14 ± 5.19 29.71 ± 0.34 3.78 ± 0.13 − 0.55 ± 0.06 0.64 ± 0.29 3 36.62 ± 0.13 5.73 ± 0.32 79.28 ± 5.64 35.54 ± 1.26 29.83 ± 0.49 3.65 ± 0.12 − 0.40 ± 0.05 0.77 ± 0.38 6 37.28 ± 0.36 6.82 ± 0.13 69.89 ± 4.24 23.18 ± 1.98 29.41 ± 0.35 3.50 ± 0.08 − 0.51 ± 0.02 0.60 ± 0.11 9 37.52 ± 0.50 6.72 ± 0.13 77.53 ± 5.70 16.17 ± 2.85 29.27 ± 0.40 3.48 ± 0.05 − 0.52 ± 0.02 0.63 ± 0.17 Fructose 0 36.10 ± 0.10 7.91 ± 0.37 84.78 ± 6.01 57.76 ± 3.89 29.17 ± 0.14 4.40 ± 0.05 − 0.36 ± 0.02 – 1 35.18 ± 0.19 7.25 ± 0.17 79.64 ± 4.25 35.07 ± 3.51 29.34 ± 0.31 4.18 ± 0.07 − 0.38 ± 0.03 0.44 ± 0.23 3 33.98 ± 0.28 6.85 ± 0.22 77.59 ± 2.95 19.23 ± 1.34 29.83 ± 0.38 4.04 ± 0.15 − 0.31 ± 0.05 1.28 ± 0.37 6 33.92 ± 0.13 7.60 ± 0.12 70.45 ± 1.32 15.03 ± 1.45 28.90 ± 0.30 3.89 ± 0.16 − 038 ± 0.05 0.62 ± 0.19 9 34.84 ± 0.23 8.15 ± 0.24 66.81 ± 3.09 10.13 ± 0.29 29.62 ± 0.49 3.88 ± 0.11 − 0.32 ± 0.08 0.54 ± 0.14 Xylitol 0 34.06 ± 0.36 7.50 ± 0.26 87.09 ± 4.61 77.62 ± 1.25 29.14 ± 0.12 4.93 ± 0.12 − 0.15 ± 0.01 – 1 34.06 ± 0.11 6.78 ± 0.18 84.90 ± 2.57 50.07 ± 0.88 29.55 ± 0.34 4.62 ± 0.12 − 0.28 ± 0.04 0.73 ± 0.27 3 36.28 ± 0.33 5.80 ± 0.26 71.98 ± 4.06 32.42 ± 0.82 29.37 ± 0.22 4.43 ± 0.07 − 0.37 ± 0.01 0.65 ± 0.28 6 35.43 ± 0.25 7.04 ± 0.05 70.15 ± 1.53 22.15 ± 2.19 29.80 ± 0.40 4.04 ± 0.02 − 0.43 ± 0.01 1.18 ± 0.35 9 36.30 ± 0.10 7.21 ± 0.19 70.70 ± 2.20 16.11 ± 1.01 29.12 ± 0.07 3.96 ± 0.03 − 0.40 ± 0.01 0.99 ± 0.39 Erythritol 0 40.60 ± 0.14 6.85 ± 0.26 79.10 ± 6.24 65.74 ± 3.23 29.16 ± 0.20 5.94 ± 0.14 − 0.37 ± 0.01 – 1 40.26 ± 0.42 6.75 ± 0.36 75.51 ± 9.02 52.85 ± 2.76 29.63 ± 0.57 5.75 ± 0.04 − 0.42 ± 0.03 0.44 ± 0.32 3 40.55 ± 0.13 6.13 ± 0.13 77.82 ± 7.43 31.17 ± 1.84 29.50 ± 0.26 5.72 ± 0.03 − 0.38 ± 0.08 0.52 ± 0.32 6 40.78 ± 0.15 7.08 ± 0.10 76.98 ± 8.73 19.29 ± 1.17 29.53 ± 0.19 5.16 ± 0.04 − 0.44 ± 0.03 0.94 ± 0.28 9 40.46 ± 0.17 6.64 ± 0.36 79.24 ± 5.37 14.25 ± 0.84 28.96 ± 0.17 5.05 ± 0.03 − 0.36 ± 0.01 0.95 ± 0.08 Mean ± standard deviation sweeteners used, followed by a gradual decrease. Statistical evaluation Thus, in the 9th month the total polyphenol content measured by Folin assay ended up in values that were even higher than the initial ones (6.72 mg GAE/g for sucrose- and 8.15 mg GAE/g for fructose-based jams) or close to the initial val- ues (7.21 mg GAE/g for xylitol- and 6.64 mg GAE/g for erythritol-sweetened jams). Results of the repeated measures ANOVA test showed that the sweetening agent (p < 0.001) and time (< 0.001) had a significant effect on the total poly- phenol content of the jams. Significantly lower values were obtained for jams containing sucrose compared to jams containing fructose and xylitol. Fructose-based samples showed also significantly higher values compared to eryth- ritol- and xylitol-based jams. Analysis of the effect of stor- age time showed that significantly lower polyphenol con- tent was obtained for samples in the 3rd month compared to all the other sampling points, while 1-month old jams showed also significantly lower phenolic content than 6- and 9-month-old samples. However, these changes did not reflect only the evolution of the original polyphenol con- tent of the jams, where a decay was registered for almost all the compounds (especially the anthocyanins) monitored by LC–MS. The chemistry behind the differences observed by the two methods involves parallel processes: in addition to the degradation of the original phenolic compounds, the total polyphenol content measured by the Folin method cov- ers other transformations as well, especially those result- ing from the Maillard reaction, where new antioxidants are formed. Therefore, Folin assay will not reflect properly the overall changes of the endogenous phenolic compounds in jams with different sweeteners. f For most of the jams, polyphenol changes were not reflected in the decay of antioxidant capacity; Pearson corre- lation coefficients revealed weak correlations, which are gen- erally not significant: sucrose: r = − 0.013, p = 0.953, fruc- tose: r = -0.405, p = 0.049 (significant at 0.05 level, 2-tailed), xylitol: r = 0.312, p = 0.129, erythritol: r = − 0.198, p = 0365. ABTS values showed a generally a non-linear decreasing 1 3 3 3 European Food Research and Technology (2020) 246:2187–2204 2193 antioxidant activity of our blackberry preparations. It is also known that fructose, as a monosaccharide, has a faster rate of Maillard reaction than sucrose, which contributes to this reaction only after its hydrolysis. Statistical evaluation This can account for the differences in antioxidant properties of sucrose- and fruc- tose-containing jams during storage, especially the rise of antioxidant capacity from month 6 to month 9 [34]. Finally, transformations of anthocyanins and other components dur- ing storage lead to polymerization and formation of new molecules that can react with Folin–Ciocalteu reagent, thus increasing the apparent polyphenol content. Hence, our results confirmed that the Folin–Ciocalteu method is not appropriate for observing polyphenol changes during stor- age and it should be definitely completed by quantitative monitoring of the native individual phenolics to follow their evolution. trend with some exceptions (Table 1). An increase of 112% was detected for sucrose from month 6 to month 9, which was not observed for the rest of the sweeteners. Results of the repeated measures ANOVA test of ABTS antioxidant capacity showed that it was not affected significantly by the sweetening agent (p = 0.158), while storage time had a sig- nificant effect on the results (p < 0.001). Samples stored for 6 and 9 months showed significantly lower ABTS values compared to the freshly prepared jam samples and samples stored for 1 month, while jams stored for 3 months showed significantly higher antioxidant capacities compared to the 6-month-old samples, and significantly lower compared to 1-month old samples. A number of papers report decay of polyphenols (spe- cifically anthocyanins) during thermal processing of fruits and vegetables [10, 31], while others found an inconsist- ent trend in the variation of these substances. Although a decrease in the antioxidant potential is typical for short heat treatments, a retrieval of these properties has been reported during prolonged heat treatment and/or storage. In our case, the observed oscillation in total polyphenol content and ABTS antioxidant activity during 9 months of storage were in line with the results obtained by many others for various products, including fruit liqueurs and diet jams [10, 32]. Many studies found that phenolic antioxidants do not fol- low a specific reaction order. Even an increase of phenolic compounds was observed in the case of several processed fruit products, while native phenolics were degraded. The apparently unpredictable fluctuation of these values was explained by multiple mechanisms, including caramelising, Strecker degradation and Maillard reaction, degradation of phenolics with higher molecular mass to smaller phenolic molecules with potential antioxidant activity and various transformations of anthocyanins. Statistical evaluation Sugar-derived products formed in the advanced stages of the Maillard reaction, specifically furfural and hydroxylmethylfurfural, which were not detected in sugar-free jams, are known to accel- erate anthocyanin degradation, leading to the formation of anthocyanin-procyanidin polymers and inducing a decay in global antioxidant capacity [33] Finally, in the last stage of the Maillard reaction brown-coloured melanoidins with high molecular weights are formed that possess antioxidant activity. On the other hand, some of the reaction products formed in the early stages of the Maillard reaction can act as pro-oxidants, thus the whole Maillard process contributes to initial decay and later an increase in antioxidant activ- ity [10], which was in line with our observations. Antioxi- dant compounds depletion in thermally treated fruits is also attributed to consumption of ascorbic acid and polyphenols as reactants in the Maillard reaction [10], ascorbic acid being also consumed in the caramelization reaction between reduc- ing sugars and ascorbic acid, which can partly explain the observed incoherence between changes in polyphenol and Additionally, the inconsistent trend in antioxidant activ- ity change can be attributed to various reasons, including the hydrolysis of the flavonoid glycosides, delivering an additional -OH moiety that can react with the ABTS cation radical [32]. Moreover, the interaction of the phenolic com- pounds or their oxidized form with the sugar molecules and the consequent formation of the reduced form of phenolic compounds that can interact with ABTS radicals [35] or— to a lesser extent—the presence of non-polyphenol antioxi- dants can also contribute to the observed lack of correlation between polyphenol content and ABTS radical scavenging activity obtained in our study. Other authors attribute the phenomenon to other factors, such as increased antioxidant power of polyphenols at an intermediate state of oxidation, increase in reducing sugar and formation of Maillard prod- ucts, known to show a high antioxidant activity, which is often exerted in mechanisms similar to that of the ABTS assay [36]. As regards the impact of the sweeteners used, no significant differences were reported in the antioxidant activity (including ABTS radical scavenging activity) of aronia jams prepared with sucrose, xylitol and erythritol, whereas—in accordance with our findings—significant differences in total polyphenol content were found, never- theless, these authors obtained the highest TPC values for xylitol [37]. Total monomeric anthocyanin content Anthocyanin degradation, one of the main processes affect- ing total phenolics content is reported by many authors in berry products and is partly attributed to indirect oxida- tion by the phenolic quinones generated by the endogenous polyphenol oxidase and peroxidase enzymes [10]. Losses of monomeric anthocyanins in berry preserves take place via both anthocyanin degradation and polymerization, even before heat treatment (by enzymatic polymerization) and during storage. Nevertheless, these polymeric compounds 1 3 European Food Research and Technology (2020) 246:2187–2204 2194 their evolution during the investigation period are shown in (Table 2). are supposed to contribute to the overall antioxidant capac- ity, and compensate degradation losses, even though their in vivo activity may be completely different. They are also reported to be resistant to colour changes, regardless of pH [7, 10]. Most of the polyphenols reported in fresh blackber- ries (Rubus fruticosus L.) were found in the jams prepared with either sugars or sugar alcohols The main polyphenols detected were flavonoids: anthocyanins (cyanidin-3-O- glucoside/kuromanin and cyanidin-rutinoside/keracyanin), flavan-3-ols (epicatechin, catechin), flavonols (quercetin, quercetin-glucoside and quercetin-3-O-rutinoside/rutin). This is in line with the values reported for blackberries, widely known for their high phenolic content. The most important polyphenols in whole fruit are anthocyanins, from which cyanidin-3-glucoside was the dominant one in blackberry jams (between 0.19 and 21.44 mg/100 g), whereas cyanidin-rutinoside was detected only in trace amounts [18]. The main phenolic acids and their deriva- tives detected in our blackberry jams were chlorogenic acid (5-CQA), neochlorogenic acid (3-CQA), p-coumaric acid and ferulic acid. These findings are in accordance with Belitz et al., mentioning hydroxycinnamic and hydroxyben- zoic acid derivatives as main non-flavonoids in blackberries: caffeoylquinic acids (chlorogenic and neochlorogenic acid) (45–53 mg/kg), 4-hydroxybenzoic acid (p-coumaric acid) (10–16 mg/kg), protocatechuic acid (68–189 mg/kg) and gallic acid (8–67 mg/kg) [13]. Their decomposition during storage reflected the main trends observed in the changes of monomeric anthocyanins, fructose being the sweetener that resulted in the quickest degradation during the whole period. Xylitol generally showed slightly better protection potential than erythritol, and both were superior to sucrose in many cases. Thus, sugar alcohols, especially xylitol, seemed to be the most suitable choice in terms of conservation of fruit- based polyphenols in the jams prepared. This was in accord- ance with the colour changes of these preparations observed on the red-green scale. Deterioration of anthocyanins was registered during the whole storage period for all the sweeteners. Total monomeric anthocyanin content Anthocya- nin content of freshly prepared sucrose (74.03 ± 3.56 mg cyanidin-3-glucoside equivalents/100 g) and xylitol (77.62 ± 1.25 mg cyanidin-3-glucoside equivalents/100 g) jams was higher than for jams with fructose (57.76 ± 3.89 mg cyanidin-3-glucoside equivalents/100 g) and erythritol (65.74 ± 3.23 mg cyanidin-3-glucoside equivalents/100 g), but these differences were partly compensated during stor- age; however, by the end of the 9th month the fructose-con- taining jam showed a lower monomeric anthocyanin content than the other samples.i Degradation of anthocyanins followed first-order kinetics, as described in previous reports on processed blackberry products [8, 10]. The reaction rate constants [k = -ln(A/ A0t)] and the half-life values [t1/2 = ln (2)/k] of the degra- dation showed that the reaction was the slowest in the case of xylitol (k = 0.0046 day−1, t1/2 = 150.68 days), followed by sucrose (k = 0.0051 day−1, t1/2 = 135.91 days) and erythritol (k = 0.0053 day−1, t1/2 = 130.78 days), while decomposi- tion was the fastest in the fructose-containing preparation (k = 0.0055 day−1, t1/2 = 126.03 days). In line with our obser- vations, in strawberry jams fructose is reported to have a more pronounced degradation effect on anthocyanins com- pared to glucose, sucrose and maltose [38]. This can be attributed to the enhanced rate of the Maillard reaction in the presence of fructose and thus to the formation of furfural and hydroxylmethylfurfural, shown to have a destructive effect on anthocyanins, as mentioned earlier. Compared to fructose, erythritol and xylitol performed better, but only xylitol was superior to sucrose in terms of a lower degradation rate of anthocyanins. In a study, sour cherry purees prepared with sugar alcohols, erythritol and xylitol also showed protective effects on anthocyanins after 6-months storage and both were superior to sucrose when preparations were stored cooled, but not when storage took place at a higher temperature [39]. In another paper, pre-pro- cess sucrose addition resulted in colour improvement. This was attributed to a stabilizing effect of sugar on monomeric anthocyanins by decreasing the rate of anthocyanin degrada- tion. This stabilizing effect is thought to be a consequence of a drop in water activity [32]. Total monomeric anthocyanin content A gradual decomposition of the main anthocyanin, cyanidin-3-glucoside was observed for all the sweeteners: its amount in the 9th month was approximately similar in sucrose- (6.1 ± 0.5 mg/100 g), xylitol- (7.3 ± 0.1 mg/100 g) and erythritol- (5.7 ± 1.3 mg/100 g) containing jams (around 25–30% of the original amounts: 31.3 ± 3.5, 26.6 ± 5.2 and 28.8 ± 0.5 mg/100 g for sucrose, xylitol and erythritol, respectively), but it was drastically reduced in fructose- based preparation (2.5 ± 0.3 mg/100 g, i.e., around 11% of the original amount of 23.3 ± 3.1 mg/100 g). The reaction rate constants [k = − ln(A/A0t)] and the half-life values (t1/2 = ln (2)/k) derived from cyanidin-3-glu- coside data were in accordance with the ones derived from the total monomeric anthocyanin measurements regard- ing fructose and xylitol; the two sweeteners resulting in the quickest and the slowest degradation of anthocyanins, respectively. Xylitol provided the slowest degradation (k = 0.0041 day−1, t1/2 = 169.06 days) and the decomposition Polyphenol profile Changes in phenolic composition during storage were moni- tored by LC–MS (Fig. 2). The individual compounds and 1 3 European Food Research and Technology (2020) 246:2187–2204 2195 2195 European Food Research and Technology (2020) 246:2187–2204 was the quickest in the fructose-containing preparation (k = 0.0075 day−1, t1/2 = 92.42 days). However, in contrary to total monomeric anthocyianin-based results, the order of erythritol (k = 0.0056 day−1, t1/2 = 123.78 days) and sucrose (k = 0.0057 day−1, t1/2 = 121.61 days) was swapped. Cyanidin-3-rutoside decomposed in a very similar man- ner to cyanidin-3-glucoside, whereas quercetin glycosides l catechin, and the former diminished most substantially in the fructose-based preparation, where its final amount was approximately half or even less (1.3 ± 0.0 mg/100 g) com- pared to the other jams (2.6 ± 0.0 mg/100 g for sucrose, 3.7 ± 0.1 mg/100 g for xylitol, 2.3 ± 0.0 mg/100 g for eryth- ritol). The observed increase in catechin content might be attributed to its formation by gradual epimerization of Fig. 2 UHPLC-ESI–MS/MS chromatogram of the freshly- prepared sucrose-containing sample. Chromatograms are showing the measured quantita- tive MRM transitions, indicated in each chromatogram next to the compound name 0 100 3-CQA; 353-179 0 100 cy-3-glu; 449-287 0 100 cy-3-rut; 595-287 0 100 catechin; 289-245 0 100 5-CQA; 353-191 0 100 caﬀeic acid; 179-135 0 100 epicatechin; 289-245 0 100 p-coumaric acid; 163-119 0 100 ferulic acid; 193-134 0 100 run; 609-300 0 100 quercen-glu; 463-271 0 100 quercen; 301-151 catechin, and the former diminished most substantially in the fructose-based preparation, where its final amount was approximately half or even less (1.3 ± 0.0 mg/100 g) com- pared to the other jams (2.6 ± 0.0 mg/100 g for sucrose, 3.7 ± 0.1 mg/100 g for xylitol, 2.3 ± 0.0 mg/100 g for eryth- ritol). The observed increase in catechin content might be attributed to its formation by gradual epimerization of epicatechin. Slight increase of some compounds, like phe- nolic acids (p-coumaric acid, caffeic acid) and a flavonoid catechin, and the former diminished most substantially in the fructose-based preparation, where its final amount was approximately half or even less (1.3 ± 0.0 mg/100 g) com- pared to the other jams (2.6 ± 0.0 mg/100 g for sucrose, 3.7 ± 0.1 mg/100 g for xylitol, 2.3 ± 0.0 mg/100 g for eryth- ritol). Polyphenol profile The observed increase in catechin content might be attributed to its formation by gradual epimerization of epicatechin. Slight increase of some compounds, like phe- nolic acids (p-coumaric acid, caffeic acid) and a flavonoid was the quickest in the fructose-containing preparation (k = 0.0075 day−1, t1/2 = 92.42 days). However, in contrary to total monomeric anthocyianin-based results, the order of erythritol (k = 0.0056 day−1, t1/2 = 123.78 days) and sucrose (k = 0.0057 day−1, t1/2 = 121.61 days) was swapped. was the quickest in the fructose-containing preparation (k = 0.0075 day−1, t1/2 = 92.42 days). However, in contrary to total monomeric anthocyianin-based results, the order of erythritol (k = 0.0056 day−1, t1/2 = 123.78 days) and sucrose (k = 0.0057 day−1, t1/2 = 121.61 days) was swapped. Cyanidin-3-rutoside decomposed in a very similar man- ner to cyanidin-3-glucoside, whereas quercetin glycosides seemed to be less sensitive to storage. Polyphenol profile Regarding flavan- 3-ols, epicatechin was far more sensitive to storage than 1 European Food Research and Technology (2020) 246:2187–2204 2196 Table 2 Phenolic compound content during the storage of jams (mg/100 g jam) Mean, standard deviation (in italics, under the corresponding mean values) S sucrose, F-fructose, X xylitol, E erythritol, the numbers following the letters denote the sampling months Compound name Transition S0 S1 S3 S6 S9 F0 F1 F3 F6 F9 X0 X1 X3 X6 X9 E0 E1 E3 E6 E9 3-CQA 353.1—> 191.1 5.9 5.4 5.3 5.1 4.9 6.3 5.3 5.0 5.1 5.0 6.9 6.6 6.0 6.6 6.4 7.5 6.4 5.6 6.0 5.6 0.4 0.3 0.1 0.2 0.2 0.6 0.3 0.1 0.2 0.2 0.8 0.3 0.1 0.3 0.3 0.2 0.4 0.2 0.3 0.3 5-CQA 353.1—> 190.9 0.2 0.2 0.20 0.2 0.2 0.2 0.3 0.2 0.2 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.2 0.2 0.3 0.3 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Caffeic acid 179.0—> 135.0 0.2 0.1 0.2 0.3 0.3 0.3 0.2 0.2 0.3 0.3 0.3 0.3 0.2 0.4 0.4 0.2 0.2 0.2 0.3 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Catechin 289.1—> 244.9 0.6 0.5 0.6 0.9 0.9 0.5 0.6 0.6 0.6 0.6 0.9 0.7 0.8 1.3 1.3 0.5 0.7 0.8 1.1 1.1 0.1 0.0 0.1 0.1 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.0 Cyanidin-glucoside 449.1—> 137.1 31.3 24.0 16.9 9.9 6.1 23.3 15.8 8.5 4.7 2.5 26.6 20.3 14.9 11.0 7.3 28.8 22.9 14.4 9.6 5.7 3.5 1.1 0.7 0.1 0.5 3.1 1.2 0.7 0.6 0.3 5.2 3.8 0.5 0.4 0.1 0.5 2.1 0.6 0.3 1.3 Cyanidin-rutinoside 595.2—> 449.0 2.6 2.3 1.6 0.8 0.5 2.0 1.6 0.8 0.4 0.2 2.4 1.9 1.4 1.0 0.6 2.1 1.7 1.2 0.7 0.5 0.4 0.4 0.2 0.1 0.1 0.5 0.1 0.1 0.0 0.0 0.4 0.1 0.2 0.1 0.0 0.5 0.3 0.0 0.1 0.0 Epicatechin 289.1—> 245.0 10.7 7.6 5.2 3.7 2.6 9.3 6.0 3.5 2.4 1.3 15.7 10.2 6.2 5.3 3.7 9.8 8.8 5.0 4.6 2.9 2.1 0.5 0.2 0.1 0.0 1.0 0.2 0.1 0.0 0.0 2.3 0.5 0.1 0.1 0.0 2.5 1.0 0.3 0.0 0.0 Ferulic acid 193.1—> 133.9 0.4 0.3 0.3 0.4 0.4 0.5 0.4 0.3 0.4 0.5 0.5 0.4 0.3 0.4 0.5 0.5 0.4 0.3 0.4 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0 p-coumaric acid 163.0—> 119.0 0.2 0.2 0.3 0.4 0.5 0.3 0.2 0.4 0.3 0.4 0.4 0.3 0.3 0.5 0.5 0.3 0.3 0.2 0.4 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Quercetin 301.0—> 150.9 1.4 1.3 1.1 1.2 1.0 1.6 1.2 1.0 1.1 1.01 1.5 1.3 0.9 1.1 1.2 0.9 1.0 0.8 1.1 1.1 0.2 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.0 0.0 0.1 Quercetin-glucoside 463.1—> 271.0 22.2 19.6 17.8 18.9 17.0 21.4 19.6 17.4 18.5 17.3 22.2 22.0 17.9 18.5 17.5 22.7 18.9 15.9 17.5 17.6 1.1 2.0 1.0 0.6 0.9 2.9 1.1 0.8 0.6 0.4 2.7 0.7 1.2 1.0 1.2 0.1 1.9 0.8 0.9 1.2 Rutin 609.1—> 300.1 4.7 4.5 4.2 4.5 4.1 5.1 4.6 4.0 4.6 4.3 5.5 5.1 4.3 4.7 4.6 4.7 4.7 3.8 4.2 4.2 0.8 0.3 0.3 0.1 0.2 0.4 0.3 0.2 0.1 0.1 0.6 0.2 0.3 0.2 0.3 1.1 0.3 0.1 0.3 0.3 Sum of phenolic compounds 80.3 65.9 53.6 46.2 38.5 70.7 55.6 41.8 38.8 33.7 83.3 69.4 53.5 51.1 44.4 78.3 66.2 48.5 46.4 40.1 Mean, standard deviation (in italics, under the corresponding mean values) S sucrose, F-fructose, X xylitol, E erythritol, the numbers following the letters denote the sampling months p p Colour changes Colour changes were monitored on the L, a and b* scales during storage time, colour differences (ΔE) were also calcu- lated (Table 1). Lightness of the jams showed an increase in the first three months and a decrease until the end of the stor- age period in case of sugars, whereas these trends ran almost inversely for sugar alcohol-containing jams, having ended up in very similar values. Results of the repeated meas- ures ANOVA test of L* values showed that the sweetening agent had no significant effect on the lightness of the jams, while the storage period produced such an effect (p = 0.03). The pairwise comparison showed no significantly different groups using the Bonferroni adjustment at p < 0.05, how- ever, the least significant difference test (that is equivalent to no adjustment) showed (p = 0.021) that the samples stored for 9 months were significantly darker than samples stored for 1 and 3 months. This is somewhat controversial to the observed increase of lightness of various strawberry jams [39] or cherry jams, as observed by Rababah et al. (2014) [41]. Mamede et al. (2013) found that sucrose-containing jams made of umbu-caja were darker than those prepared with xylitol [40], which was not the case for our blackberry jams. It was reported that sucrose-containing aronia jams showed the lowest L, a and b* values, followed by xylitol and erythritol [37]—this was confirmed in this study for a* and b* values. Some other authors also found that the intensity of red hue was higher in light cherry jams com- pared to those with higher sucrose content, both at prepa- ration and after storage [42]. The positive effect of xylitol on colour reported for strawberry jams [43] and the pro- tecting effect of both xylitol and erythritol on the colour of sour cherry purees [39] was confirmed in our experiments. Erythritol-containing jams were significantly higher in red hue than all the other jams, suggesting that total concen- tration of monomeric anthocyanin species are not the only determinant in colour expression. This phenomenon was also reported by Garcia-Viguera et al. (1997) [15] and oth- ers [10] and was explained by the co-pigmentation: phenolic acids, flavonols and flavan-3-ols are known to interact with anthocyanins, producing an enhancement of colour intensity. Ifie et al. Mean, standard deviation (in italics, under the corresponding mean values) S sucrose, F-fructose, X xylitol, E ery 1 3 European Food Research and Technology (2020) 246:2187–2204 2197 aglycone (quercetin, in case of erythritol-based jams) was noticed during storage. This can be explained either by matrix decomposition processes, that may result in the lib- eralisation of smaller free phenolic acids from more complex native molecules, e.g., p-coumaric acid from chlorogenic acids, or the release of free flavonoid aglycone forms from the corresponding glycosides (e.g., quercetin from rutin). of the sweetening agent on the bluishness of the samples (p < 0.001), while storage time did not affect significantly the results (p = 0.094). Significant difference was found between erythritol sweetened jams compared to the sucrose-, fruc- tose- and xylitol-sweetened jams: erythritol-sweetened jams were less bluish than the other three jams. ΔE values showed that the global colour differences compared to the freshly prepared jams are higher at the end of the storage period for sugar alcohol-containing jams (0.99 ± 0.39 for xylitol and 0.95 ± 0.08 for erythritol). These differences are almost double compared to sugar-containing jams (0.63 ± 0.17 for sucrose and 0.54 ± 0.14 for fructose), however, repeated measures ANOVA revealed no significant differences determined by either sweetener or time. It should be noted that ΔE values are not following a continuously increasing trend, thus reflecting the fluctuating changes of L, a and b* values. This way, the maximum ΔE values are not necessarily reached by the end of the storage period. Nonetheless, in all the cases these values denote colour dif- ferences that are not perceived by humans (ΔE < 1) or, in the case of xylitol-based jams, perceived only by experienced observers (1 < ΔE < 2) [41]. Sensory analysis During the 9-months storage period, the sensory proper- ties of our blackberry jams were comparatively evaluated by the panel. Sensory profile of the samples at the beginning and at the end of the period investigated are shown in Fig. 1. In addition to colour, changes in the phenolic profile are also linked to other sensory attributes of the products, their alteration may be accompanied by changes in flavour and aroma as well. There are many papers reporting a compari- son of sensory properties of preserved products containing sugar or sugar alcohols, especially xylitol. This latter is found to show similar or even superior properties in various food products when compared to sucrose. Sensory proper- ties of umbu-caja jams made with sucrose were close to the ones made with xylitol, no significant differences were obtained for appearance, flavour and texture [40]. During the comparative sensory evaluation of jams containing sucrose, xylitol or erythritol, the xylitol-containing jam reached the best results in terms of taste, flavour and overall accept- ance [35]. Xylitol showed a good performance with other According to the two-way repeated-measures ANOVA analysis (Table 3), the sweetening agents had a significant effect (significance level p < 0.01) on all the investigated sensory parameters. Erythritol-sweetened jams were sig- nificantly more reddish (higher hue values) compared to the other groups, while sucrose-sweetened jams were charac- terized by significantly lower-rated hues, i.e. hues closer to black. A remarkable consistency was obtained between the evaluation of the panel for the hue (black-dark red scale) and the results obtained by colorimetry for a* values: erythritol was found to show the most intense red colour, which is reportedly one of the most important attributes, significantly determining consumer preference [47]. On the other hand, Table 3 Significant differences between physicochemical and sensory attributes as calculated by repeated measures ANOVA regarding sweeten- ing agent and storage time pre-set reference values Mean ± standard deviation. Colour changes found that hydroxycinnamic acids are involved in the formation of new stable anthocyanin-derived pigments through condensation reactions with anthocyanins which may account for maintaining colour intensity [44]. Com- paring the concentration of the phenolic compounds men- tioned above in sucrose- and erythritol-containing jams, it was stated that all of them (i.e., quercetin derivatives: rutin, quercetin glucoside, quercetin, flavan-3-ols: catechin, epi- catechin and all the phenolic acids (5-CQA, 3-CQA, p-cou- maric acid, ferulic acid)) were present in higher amounts in the jams containing erythritol than in the jams containing sucrose, contributing thus to colour compensation. Never- theless, a strong correlation between a (red) values and Results of the repeated measures ANOVA test of a* val- ues showed that both sweetening agent and storage time had a significant effect on the red hue of the samples at p < 0.001 significant level, according to the within-subject effect tests. In the case of the effect of sweetening agents, significant differences were found among all of the groups, the decreas- ing order being: erythritol, xylitol, fructose, sucrose. The effect of the storage time showed that samples stored for 6 and 9 months showed significantly lower a* values than the others and samples stored for 9 months obtained also signifi- cantly lower a* compared to the 6 months stored samples, i.e. the red hue of the samples was significantly reduced from month 6. On the other hand, 1-month old samples, showed a significant decrease in redness compared to the fresh samples, while these differences were statistically not relevant for 3-month old samples. Changes on the yellow-blue scale are inconsistent, as a result, no marked decrease was observed in the global b* values at the end of the observation period, the initial dif- ferences having been equalized. Results of the repeated measures ANOVA test of b* showed a significant effect 1 3 European Food Research and Technology (2020) 246:2187–2204 2198 total monomeric anthocyanin content was observed for all the sweeteners (Pearson correlation coefficients at p = 0.05 are: sucrose: 0.98, fructose: 0.99, xylitol: 0.97, erythritol: 0.92). fruit-containing products as well, reaching higher scores for fruity notes than sucrose [45]. The use of xylitol led to a good sensory acceptance even at high concentrations [46] being thus generally considered a good choice as a sugar substitute in jams. Sensory analysis Superscript letters show significant differences (p < 0.05) between jams for different sweeteners and storage time: capital letters refer to sweetener models lowercase letters refer to storage time models Parameter Sucrose Fructose Xylitol Erythritol Month 0 Month 1 Month 3 Month 6 Month 9 TPC 6.38 ± 0.48A 7.55 ± 0.52C 6.86 ± 0.62B 6.69 ± 0.4AB 7.20 ± 0.62bc 6.72 ± 0.48b 6.13 ± 0.51a 7.13 ± 0.31c 7.18 ± 0.66c ABTS 78.29 ± 6.25A 75.85 ± 7.46A 76.97 ± 8.13A 77.73 ± 6.98A 84.98 ± 4.22d 82.27 ± 3.38c 77.14 ± 4.84bd 70.85 ± 2.75a 70.79 ± 5.71ab L* 29.49 ± 0.41A 29.51 ± 0.57A 29.39 ± 0.35A 29.34 ± 0.50A 29.22 ± 0.28ab 29.56 ± 0.40b 29.67 ± 0.62b 29.47 ± 0.44ab 29.24 ± 0.39a a* 3.68 ± 0.22A 4.08 ± 0.23B 4.40 ± 0.41C 5.54 ± 0.39D 4.82 ± 0.77d 4.57 ± 0.75c 4.49 ± 0.86 cd 4.15 ± 0.64b 4.09 ± 0.60a b* − 0.46 ± 0.19A − 0.35 ± 0.06A − 0.3 ± 0.16A 0.01 ± 0.09B − 0.21 ± 0.23a − 0.22 ± 0.31a − 0.27 ± 0.18a − 0.35 ± 0.18a − 0.32 ± 0.17a ΔE 0.66 ± 0.25A 0.76 ± 0.41A 0.92 ± 0.34A 0.99 ± 1.24A – 0.85 ± 1.26a 0.78 ± 0.40a 0.85 ± 0.32a 0.84 ± 0.32a Sensory attributes Hue 20A 24.7 ± 7.2B 27.2 ± 9.5B 38.3 ± 15.7C 22.6 ± 7.4a 29.1 ± 14.1ab 27.4 ± 12.4ab 32.4 ± 13.8b 26.5 ± 8.3ab General odour intensity 60A 64.4 ± 9.9B 58.8 ± 10.3A 56.4 ± 13.1A 62.6 ± 13.1a 58.4 ± 8.8a 60.9 ± 7.1a 59.6 ± 11.7a 57.8 ± 8.0a Black- berry odour 60BC 63.7 ± 10.1C 56.7 ± 11.6AB 56.0 ± 9.4A 61.0 ± 8.7ab 55.6 ± 10.9a 60.3 ± 9.4b 58.5 ± 10.6ab 59.6 ± 6.3ab Sweet odour 30A 37.4 ± 14.4B 36.2 ± 11.5B 31.4 ± 7.7AB 36.7 ± 12.6a 32.4 ± 7.3a 34.6 ± 11.3a 33.5 ± 10.5a 31.6 ± 9.1a Stickiness 40A 44.8 ± 11.6AB 46.4 ± 12.2B 46.0 ± 15.9B 45.3 ± 10.8b 45.6 ± 10.4b 47.1 ± 16.1ab 44.5 ± 10.8ab 39.1 ± 8.3a General taste inten- sity 60B 62.3 ± 10.2B 60.2 ± 11.4B 53.2 ± 13.9A 58.1 ± 10.1a 58.9 ± 10.0a 62.1 ± 10.6a 59.4 ± 13.4a 56.2 ± 9.1a Black- berry flavour 55B 58.3 ± 9.1C 56.0 ± 8.4BC 45.1 ± 13.1A 54.5 ± 6.4b 53.5 ± 11.4ab 57.5 ± 12.5b 49.7 ± 11.0a 52.7 ± 7.7ab Sweet taste 40A 46.5 ± 12.5B 45.7 ± 12.0AB 48.1 ± 16.7B 46.9 ± 16.4a 44.4 ± 11.4a 47.3 ± 12.5a 42.7 ± 11.9a 44.0 ± 7.9a Sour taste 40*A 48.3 ± 12.4BC 49.3 ± 14.9C 41.0 ± 12.4AB 47.2 ± 11.4a 45.5 ± 12.2a 43.4 ± 13.6a 46.2 ± 12.2a 41.0 ± 10.6a Table 3 Significant differences between physicochemical and sensory attributes as calculated by repeated measures A ng agent and storage time Mean ± standard deviation. Sensory analysis Finally, the use of sugar alcohols resulted in jams that were stickier than the sucrose-containing samples.f SSSSSSSSS E E E E E E E E E E E E F F F F F F F F F F F F X X X X X X X X X X X X -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 F2 (9.33 %) F1 (14.03 %) a a M0 M1 M3 M6 M9 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 F2 (9.33 %) F1 (14.03 %) b b b Regarding the effect of the storage time, only hue, black- berry odour and flavour, and stickiness went through signifi- cant changes (p < 0.05). Samples stored for 6 months were significantly more reddish compared to the fresh samples. Moreover, samples stored for six months were significantly weaker in blackberry flavour compared to 1- and 3-month- old samples. Jams stored for 1 month were perceived as hav- ing less intense blackberry odour compared to 3-month-old jams. Panellists in our study responded in the open-ended text fields that the erythritol samples showed a grainy, sandy texture. In addition, the samples at the endpoint were sig- nificantly less sticky than the fresh jams and jams stored for 1 month. Fig. 3 Multiple factor analysis scores plot (a) and table coordinate plot (b). E erythritol, S sucrose, F fructose, X xylitol, M denotes months, while numbers after M denote the number of months elapsed from preparation samples showed different sensory properties. Figure 3b pre- sents the connections among the different data sets. In a similar manner to Fig. 3a, clear trend can be seen along F1, meaning that sensory attributes changed not only among samples but during storage, too. Although there are many reports available on the use of electronic nose and tongue in quality monitoring of fruits and processed fruit products [48, 49], there are only a few reports discussing experiments on jams, most of the papers focusing on fruit juices or nectars [50, 51]. Generally, these tools showed good performances in differentiating variously processed fruit products, including thermal processing. Elec- tronic tongue shows better performance in most of the cases. To identify specific patterns in our sensory data matrix, multiple factor analysis (MFA) was applied (Fig. 3). Sensory analysis Superscript letters show significant differences (p < 0.05) between jams for different sweeteners and storage time: capital letters refer to sweetener models, lowercase letters refer to storage time models 1 3 3 European Food Research and Technology (2020) 246:2187–2204 2199 SSSSSSSSS E E E E E E E E E E E E F F F F F F F F F F F F X X X X X X X X X X X X -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 F2 (9.33 %) F1 (14.03 %) M0 M1 M3 M6 M9 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 F2 (9.33 %) F1 (14.03 %) a b Fig. 3 Multiple factor analysis scores plot (a) and table coordinate plot (b). E erythritol, S sucrose, F fructose, X xylitol, M denotes months, while numbers after M denote the number of months elapsed from preparation jams prepared with sugar alcohols obtained significantly lower scores for general odour intensity and blackberry odour than those containing fructose. Both fructose- and sucrose-based samples were superior to erythritol-sweetened jams in terms of blackberry odour. Fructose-containing samples were also superior to sucrose-sweetened samples in terms of their blackberry flavour. Erythritol-containing samples were inferior to the rest of the samples regarding general taste intensity and blackberry flavour. The low val- ues obtained by erythritol for blackberry flavour and gen- eral taste intensity are in accordance with those reported by Hwang et al. (2014), who did not recommend erythritol to be used as sole sweetener, because it influenced the sensory properties negatively [37]. However, erythritol- and fruc- tose-containing samples were sweeter than sucrose-based jams. The differences detected among the organoleptic prop- erties of our blackberry jams did not confirm entirely the lit- erature reporting no significant sensory differences between sugar- and xylitol-based fruit products [40]. Fructose- and xylitol-containing samples had a more intensive sweet odour than the sucrose-containing ones. On the other hand, xylitol- sweetened jams were perceived as being significantly sourer than erythritol- and sucrose-based jams, and while sucrose- containing samples were less sour than the fructose-con- taining ones. 1 3 Sensory analysis MFA performs individual PCAs on the data matrices obtained from the five sensory evaluation sessions. Figure 3a pre- sents the scores plot of the MFA containing the results of all the sessions. Erythritol-containing samples are located on the left side of the plot and are not overlapped by fruc- tose or sucrose-containing samples. Xylitol-based samples overlap with all other samples, while fructose samples are clearly different from erythritol-sweetened samples. A clear left–right differentiation is seen on Fig. 3a along with F1, in which colour, global odour and taste intensity proved to be the major contributors from the five data sets. Based on these results, we conclude that fructose- and erythritol-based In line with literature reports, linear discriminant analysis (LDA) performed on electronic tongue results (Fig. 4) pro- vided a correct classification (100%) for each group of the freshly prepared jams (month 0) according to their sweeten- ing agent. The same correct classification was obtained at the end of the storage period (100%). Distances show that sugar-based preparations are more similar to each other than 1 3 European Food Research and Technology (2020) 246:2187–2204 2200 u op a ood a a d o ogy ( 0 0) 6: 8 Linear discriminant analysis (LDA) performed on electronic tongue results in month 0 (a) and month 9 (b) Fig. 4 Linear discriminant analysis (LDA) performed on electronic tongue results in month 0 (a) and month 9 (b) to sweetener type provided average recognition and pre- diction abilities of 63.97% and 36.42%, respectively, in month 0. Measurement points of samples with erythritol or fructose were misclassified into all the other groups; points of the sample with xylitol were misclassified as the sample groups containing fructose and erythritol, while sucrose- based jams were misclassified only into the xylitol-based class. For month 9 the average recognition and predic- tion abilities were 71.60% and 40.98%, respectively. The those with sugar-alcohols. Due to the drift correction, discri- minant analysis according to the sweetener can be done for the whole storage period, this leading again to a fully correct classification [52, 53]. i On the other hand, LDA of the results obtained by the electronic nose (Fig. 5) provided no differentiation accord- ing to the sweetener used, this suggesting differences rather in taste than in the odour of the jams prepared with different sweeteners. Acknowledgements Open access funding provided by Semmelweis University. Conclusions Evaluation of blackberry jams prepared with different sweet- eners (sucrose, fructose, xylitol, erythritol) during 9-months storage revealed a series of significant differences in terms of antioxidant properties, polyphenol content, colour and sensory attributes. Thus, the type of sweetener had a measur- able impact on the evolution of both sensory attributes and antioxidant properties. Storage time also had a well-defined effect on sensory and compositional properties of jams. Funding Supported by the Doctoral School of Food Science, Szent István University. Supported by the ÚNKP-19-3-I-SZIE-71 (Zs.B.) and ÚNKP-19-4 (LA, ZK) New National Excellence Program of the Ministry for Innovation and Technology. Supported by the Janos Bol- yai Scholarship of the Hungarian Academy of Sciences (LA, ZK). LA acknowledges the support EFOP-3.6.3-VEKOP-16-2017-00005 and VEKOP-2.3.3-15-2017-00022 grants of the European Union co- financed by the European Social Fund. AG was supported by the Pre- mium Postdoctoral Researcher Program of the Hungarian Academy of Sciences. f Antioxidant capacities and total polyphenol content did not change according to a consistent trend during the inves- tigation period, this was most probably attributed to the for- mation of Maillard reaction products and polymeric com- pounds. Due to the complex processes occurring, changes in total polyphenol content measured by Folin–Ciocalteu assay were not in accordance with the decay of the indi- vidual native phenolic compounds determined by LC–MS, therefore, it was concluded that the Folin assay cannot be considered as an efficient tool in monitoring polyphenol degradation during storage of jams. Regarding anthocyanin degradation, fructose proved to produce the highest decom- position rate, while the reaction was the slowest in xylitol- containing jams. Sensory analysis Classification models built according 1 3 1 3 European Food Research and Technology (2020) 246:2187–2204 2201 220 European Food Research and Technology (2020) 246:2187–2204 results obtained showed a serious misclassification for misclassified as erythritol (12 36%) and xylitol (25 09% Fig. 5 Linear discriminant analysis (LDA) performed on electronic nose results in month 0 (a) and month 9 (b) Fig. 5 Linear discriminant analysis (LDA) performed on electronic nose results in month 0 (a) and month 9 (b) misclassified as erythritol (12.36%) and xylitol (25.09%) sample groups. results obtained showed a serious misclassification for sample group with erythritol, being classified as fructose- (71.37%) and xylitol-sweetened (28.63%) sample groups. Fructose-containing jams were misclassified as 11% belonging to xylitol-containing class, while xylitol was misclassified into all the other groups, and sucrose was Fructose-containing jams were misclassified as 11% belonging to xylitol-containing class, while xylitol was misclassified into all the other groups, and sucrose was 1 3 1 3 1 3 2202 European Food Research and Technology (2020) 246:2187–2204 Author contributions CsB drafted the manuscript, performed antioxi- dant measurements and coordinated the study, ZB and VTM executed laboratory measurements, ZB performed statistical calculations, Z. Kókai and AG performed sensory profile analysis and contributed to statistical calculations, ID performed electronic nose measurements, Z. Kovacs performed electronic tongue measurements, LA performed LC–MS measurements and made extensive revisions on the manu- script. All the authors have reviewed and approved the final article. Code availability Not applicable. Code availability Not applicable. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Compliance with ethical standards Conflicts of interest The authors declare that they have no conflict of interest. Compliance with ethics requirements This article does not contain any studies with human participants or animals performed by any of the authors. Availability of data and material Not applicable. Availability of data and material Not applicable. Colorimetric measurements revealed that erythritol-con- taining jams were significantly higher in red hue than all the other studied jams. Similarly, when compared by a trained sensory panel, the jams sweetened with erythritol were per- ceived as having been the most reddish. These findings sug- gest that anthocyanin concentration per se is not the only significant determinant of the characteristic reddish colour of blackberry jams. It is noted, however, that erythritol-con- taining jams obtained the lowest intensity scores for general taste intensity and blackberry flavour. On the other hand, fructose reached positive results in general odour intensity and blackberry flavour. Unlike the electronic nose, electronic tongue produced a correct classification according to the type of sweetener and time, this qualifying it as a potential tool applicable in the food industry for monitoring the sen- sory properties of fruit jams with different sweeteners, as well as the alteration of these characteristics during storage.f Code availability Not applicable. j p 4. Williamson G (2017) The role of polyphenols in modern nutrition. Nutr Bull 42:226–235 References Taking into consideration the positive effects that xylitol showed on anthocyanin decay, as well as its sensory scores that were not significantly lower than those obtained for sucrose, it can be concluded that xylitol may be eligible as a good alternative for diet jams. 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https://openalex.org/W2769519820	https://www.econstor.eu/bitstream/10419/176470/1/10.1186_s40854-017-0080-y.pdf	English	null	Optimal stopping investment in a logarithmic utility-based portfolio selection problem	Financial innovation	2,017	cc-by	5,743	Article Optimal stopping investment in a logarithmic utility-based portfolio selection problem Financial Innovation Provided in Cooperation with: Springer Nature Suggested Citation: Li, Xun; Wu, Xianping; Zhou, Wenxin (2017) : Optimal stopping investment in a logarithmic utility-based portfolio selection problem, Financial Innovation, ISSN 2199-4730, Springer, Heidelberg, Vol. 3, Iss. 28, pp. 1-10, https://doi.org/10.1186/s40854-017-0080-y Suggested Citation: Li, Xun; Wu, Xianping; Zhou, Wenxin (2017) : Optimal stopping investment in a logarithmic utility-based portfolio selection problem, Financial Innovation, ISSN 2199-4730, Springer, Heidelberg, Vol. 3, Iss. 28, pp. 1-10, https://doi.org/10.1186/s40854-017-0080-y Li, Xun; Wu, Xianping; Zhou, Wenxin Article Optimal stopping investment in a logarithmic utility-based portfolio selection problem Financial Innovation Provided in Cooperation with: Springer Nature Suggested Citation: Li, Xun; Wu, Xianping; Zhou, Wenxin (2017) : Optimal stopping investment in a logarithmic utility-based portfolio selection problem, Financial Innovation, ISSN 2199-4730, Springer, Heidelberg, Vol. 3, Iss. 28, pp. 1-10, https://doi.org/10.1186/s40854-017-0080-y Li, Xun; Wu, Xianping; Zhou, Wenxin Article Optimal stopping investment in a logarithmic utility-based portfolio selection problem Financial Innovation Provided in Cooperation with: Springer Nature Suggested Citation: Li, Xun; Wu, Xianping; Zhou, Wenxin (2017) : Optimal stopping investment in a logarithmic utility-based portfolio selection problem, Financial Innovation, ISSN 2199-4730, Springer, Heidelberg, Vol. 3, Iss. 28, pp. 1-10, https://doi.org/10.1186/s40854-017-0080-y Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. 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Abstract Background: In this paper, we study the right time for an investor to stop the investment over a given investment horizon so as to obtain as close to the highest possible wealth as possible, according to a Logarithmic utility-maximization objective involving the portfolio in the drift and volatility terms. The problem is formulated as an optimal stopping problem, although it is non-standard in the sense that the maximum wealth involved is not adapted to the information generated over time. Methods: By delicate stochastic analysis, the problem is converted to a standard optimal stopping one involving adapted processes. Keywords: Optimal stopping, Path-dependent, Stochastic differential equation (SDE), Time-change, Portfolio selection Keywords: Optimal stopping, Path-dependent, Stochastic differential equation (SDE), Time-change, Portfolio selection Keywords: Optimal stopping, Path-dependent, Stochastic differential equation (SDE), Time-change, Portfolio selection Optimal stopping investment in a logarithmic utility-based portfolio selection problem Xun Li1* , Xianping Wu2 and Wenxin Zhou1 Correspondence: malixun@polyu.edu.hk 1Department of Applied Mathematics, The Hong Kong Polytechnic University, Hong Kong, China Full list of author information is available at the end of the article Financial Innovation Li et al. Financial Innovation (2017) 3:28 DOI 10.1186/s40854-017-0080-y © 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 Correspondence: malixun@polyu.edu.hk 1Department of Applied Mathematics, The Hong Kong Polytechnic University, Hong Kong, China Full list of author information is available at the end of the article Background Optimal stopping problems, a kind of dynamic optimization problems allowing investors to stop investment any time before the maturity in order to maximize their profits or minimize their costs, are of great interest and of importance in various fields such as science, engineering, economics, management, and particularly in financial investment. In reality, choosing a proper time point to stop investment is of importance to hedge risk and to realize maximum return for investors. In practice, it is extremely hard to find the point at which the realized return is maximized, and therefore the investor tries to sell at a price which is as close to the maximum as possible. To help determine this time point, researchers have made significant effort toward the theory of optimal stopping, and Shiryaev et al. (2008) is one of the typical representatives along this line of research. In the field of mathematical finance, furthermore, optimal stopping has been extensively studied for pricing American-style options, which allow option holders to exercise the options before or at the maturity. The theory of optimal stopping developed in pricing American options can be further applied to determine an optimal stopping point so as to maximize return from financial Li et al. Financial Innovation (2017) 3:28 Page 2 of 10 investment for economic agents. Nevertheless, it is extremely hard to let investors realize the highest return, and therefore, the objective is to minimize the distance between the time point at which the investment is stopped and that at which the maximum return can be realized. For example, Ceci and Bassan (2004) study the mixed optimal stopping and stochastic control problems with semicontinuous final reward for diffusion processes and give some properties of the value function. Dayanik and Karatzas (2003) investigate the optimal stopping problems for one dimensional diffusions and showed how to reduce the discounted optimal stopping problem for an arbitrary diffusion process to an undis- counted one for standard Brownian motion. Choi et al. (2004) study an investor’s decision to switch from active portfolio management to passive management and modelled it to a mixture of a consumption-portfolio selection problem and an optimal stopping prob- lem. Chang et al. (2009) consider the optimal stopping problem for stochastic differential equations with random coefficients. Shiryaev et al. (2008) address the optimal stopping issue in an equity market by considering a log-normal price process. Background The mean-variance approach originated by Markowitz (1952, 1959) has been a cor- nerstone of asset allocation, investment analysis and risk management. In this literature, Merton’s (1969, 1971, 1973) seminal work is considered a benchmark on continuous- time portfolio selection. The single period model is extended by Li and Ng (2000) for multi-period case and developed by Zhou and Li (2000) for continuous-time one, respec- tively. The work of Li and Zhou (2006) reveals the high opportunity of a Markowitz mean-variance strategy hitting the expected return target before the maturity date. Nat- urally, investors also hope to decide when to stop the investment over a given investment horizon so as to maximize their profits. This idea has been further developed to deter- mine the optimal selling time for one stock by Shiryaev et al. (2008), who determined a time point at which investors can sell risky assets as close to the maximum return as possible. This again highlights the efficiency of the mean-variance analysis in the field of investment and portfolio selection. Naturally, an investor also hopes to know the time point to stop the investment over a given investment horizon so as to maximize the profit. In this paper, we devote to choosing an optimal point at which an investor stops the investment among multi assets for gaining maximum benefit. The investor is expected to maximize her personal utilities and to minimize the difference between the realized return at the stopping point and her potentially maximum return. Compared with the work of Shiryaev et al. (2008), we consider the utility function of a quadratic form instead of a relative error criterion. And since multi financial assets are considered, the drift and volatility terms involve the portfolio. These make our analysis more realistic and meaningful. The rest of the paper is organized as follows. In “Formulation” section, we formu- late the problem to a two-stage problem in which the investor is expected to maximize her personal utilities and to minimize the difference between the realized return/wealth at the stopping point and her potentially maximum return/wealth. We first derive the optimal portfolio of the sub-problem by stochastic controls methodology. Then substi- tuting the optimal portfolio into the dynamic systems, we have the optimal stopping time problem with the wealth process without the control variable in the drift and volatility terms. Background Using time-change technique, the nonadapted problem is transformed into a stan- dard optimal stopping problem. Numerical examples are presented in “Methods” section Li et al. Financial Innovation (2017) 3:28 Page 3 of 10 to demonstrate the theoretical results. “Results and discussion” section 4 concludes the work. Some technique details are relegated to an Appendix. Formulation Throughout this paper (, F, P, {Fs}s≥0) is a fixed filtered complete probability space on which defined a standard Fs-adapted m-dimensional Brownian motion {W(s), s ≥0} with W(0) = 0, and T > 0 is given and fixed, representing the terminal time of an investment horizon. There is a financial market in which m+1 securities (or assets) are traded continuously. One of the securities is a risk-free asset, whose price follows dS0(t) = rS0(t)dt, t ≥0, S0(0) = s0 > 0, (1) (1) where r > 0 is the interest rate. The other m securities are risky assets, whose prices follows where r > 0 is the interest rate. The other m securities are risky assets, whose prices follows ⎧ ⎪⎨ ⎪⎩ dSi(t) = Si(t) bidt + m j=1 σijdW j(t) , t ≥0, Si(0) = si > 0, i = 1, 2, · · · , m, (2) (2) (2) where b := (b1, b2, · · · , bm)′ is the appreciation rate, σ := (σij)m×m is the volatility, and σ ′σ is positive definite. Consider an agent, with an initial endowment x0 > 0 and whose total wealth at time s ∈[ 0, ˆT] is denoted by x(s). Assume that the trading of shares is self-financed and takes place continuously, and that transaction cost and consumptions are ignored. Then x(·) satisfies ⎧ ⎪⎨ ⎪⎩ dx(s) = rx(s) + m i=1 (bi −r)πi(s) ds + m j=1 m i=1 σijπi(s)dW j(s), 0 ≤s ≤ˆT, x(0) = x0, (3) (3) (3) where πi(s), i = 1, 2 · · · , m, denotes the total market value of the agent’s wealth in the i-th stock. We call the process π(s) := (π1(s), π2(s), · · · , πm(s))′ a portfolio of the agent. Define the running maximum wealth process where πi(s), i = 1, 2 · · · , m, denotes the total market value of the agent’s wealth in the i-th stock. We call the process π(s) := (π1(s), π2(s), · · · , πm(s))′ a portfolio of the agent. where πi(s), i = 1, 2 · · · , m, denotes the total market value of the agents wealth in the i-th stock. We call the process π(s) := (π1(s), π2(s), · · · , πm(s))′ a portfolio of the agent. Define the running maximum wealth process i-th stock. Formulation We call the process π(s) := (π1(s), π2(s), · · · , πm(s)) a portfolio of the agent. Define the running maximum wealth process Define the running maximum wealth process M(s) = max 0≤u≤s x(u), s ≥0. M(s) = max 0≤u≤s x(u), s ≥0. Assume that an investor can stop investment at any point before a pre-specified date T > 0. The question is to choose an optimal portfolio and to determine the right time to stop investment. The main objective of this study is to determine conditions for which the investor should sell her shares. Ideally, the investor would like to exit when the value is highest, which is at time s, such that x(s) = αM(T). More generally, the investor may have an investment target that is a fraction of (or possibly equal to) the maximum value, αM(T), where 0 < α ≤1. With this objective, we assume that the investor chooses an exit time to minimize the mean squared difference between exit value and investment target value. We formulate it to the following optimal stopping problem: min 0≤ˆτ≤T E x(ˆτ) −αM(T) 2 , (4) subject to ⎧ ⎨ ⎩ max π(·) E ln(x(T)) , subject to (x(·), π(·)) satisfy (3). (5) (4) (5) Li et al. Financial Innovation (2017) 3:28 Page 4 of 10 Note that the above two-stage problem setting is very insightful. It is more realistic than those addressed in Shiryaev et al. (2008) since m-dimensional financial assets are considered and the drift and volatility terms involving the portfolio. Methods Before further developing techniques derived in Shiryaev et al. (2008), we know the optimal portfolio of sub-problem (5) via stochastic control method ˆπ(s) ≡( ˆπ1(s), ˆπ2(s), · · · , ˆπm(s))′ = (σσ ′)−1(b −r1)x(s), (6) (6) ere 1 = (1, 1, · · · , 1)′ is an m-dimensional column vector. where 1 = (1, 1, · · · , 1)′ is an m-dimensional column vector. Substituting (6) into (5) yields the wealth process x(·) without the control variable in the drift and volatility terms dx(s) = x(s) (r + \|θ\|2)ds + θ′dW(s) , x(0) = x0, (7) (7) where θ = σ −1(b −r1). This is similar to the case in Shiryaev et al. (2008), but it is more mathematically complex. By virtue of a time-change technique, there exists a one-dimensional standard Brownian motion B(s), s ≥0, on (, F, P) such that θ′W(s) = B(β(s)), 0 ≤s ≤ˆT, where β(s) := \|θ\|2s. where β(s) := \|θ\|2s. Set t := \|θ\|2s, Eq. (7) is equivalent to Set t := \|θ\|2s, Eq. (7) is equivalent to Set t := \|θ\|2s, Eq. (7) is equivalent to dx(t) = x(t) {μdt + dB(t)} , (8) dx(t) = x(t) {μdt + dB(t)} , x(0) = x0, (8) (8) dx(t) = x(t) {μdt + dB(t)} , x(0) = x0, (8) where μ = r \|θ\|2 + 1. Thus, the problem (4) is equivalent to where μ = r \|θ\|2 + 1. Thus, the problem (4) is equivalent to where μ = r \|θ\|2 + 1. Thus, the problem (4) is equivalent to where μ = r \|θ\|2 + 1. Thus, the problem (4) is equivalent to min 0≤τ≤T E x(τ) −αM(T) 2 (9) (9) over τ ∈T , the set of all Ft-stopping time τ ∈[ 0, T], where T = \|θ\|2T. Consequently, the value function associated with problem (9) is over τ ∈T , the set of all Ft-stopping time τ ∈[ 0, T], where T = \|θ\|2T. Consequently, the value function associated with problem (9) is V(t, x, M) = min t≤τ≤T E (x(τ) −αM(T))2\|Ft = min t≤τ≤T E x(τ)2 −2αx(τ)M(T) + α2M(T)2\|Ft = min t≤τ≤T E x(τ)2 −2αx(τ)E[ M(T)\|Fτ] +α2E[ M(T)2\|Fτ] Ft . (10) (10) Defining ν := μ −1 2, we rewrite x(t) := x(0) exp(νt + B(t)), M(t) := x(0) exp max 0≤u≤t(νu + B(u)) . Li et al. Methods t follo s (10) that It follows (10) that V(t, x, M) = min t≤τ≤T E x(τ)2−2αx(τ)ψ(τ, x(τ), M(τ))+α2φ(τ, x(τ), M(τ))\|Ft , (13) which is governed by which is governed by ⎧ ⎪⎨ ⎪⎩ max L V, V −x2 + 2αxψ −α2φ = 0, VM(t, M, M) = 0, V(T, x, M) = (x −αM)2, ⎧ ⎪⎨ ⎪⎩ max L V, V −x2 + 2αxψ −α2φ = 0, VM(t, M, M) = 0, V(T, x, M) = (x −αM)2, (14) (14) ⎨ ⎪⎩V(T, x, M) = (x −αM)2, where the operator L is defined by Methods Financial Innovation (2017) 3:28 Page 5 of 10 Denote ψ(t, x(t), M(t)) = E[ M(T)\|Ft] and φ(t, x(t), M(t)) = E[ M(T)2\|Ft]. Then Denote ψ(t, x(t), M(t)) = E[ M(T)\|Ft] and φ(t, x(t), M(t)) = E[ M(T)2\|Ft]. Then ψ(t, x(t), M(t)) = E[ M(T)\|Ft] = E x(0) exp max 0≤u≤T(νu + B(u)) Ft = E x(0) exp max max 0≤u≤t(νu + B(u)), max t≤u≤T(νu + B(u)) Ft = E x(0) exp max max 0≤u≤t(νu + B(u)), (νt + B(t)) + max 0≤u≤T−t(νu + B(u)) Ft = E x(t) exp max max 0≤u≤t(νu + B(u)) −(νt + B(t)), max 0≤u≤T−t(νu + B(u)) Ft = E x(t) exp max y, max 0≤u≤T−t(νu + B(u)) y = max 0≤u≤t(νu + B(u)) −(νt + B(t)) = x(t)G1 t, ln M(t) x(t) , (11) (11) where G1(t, y) = E exp max y, max 0≤u≤T−t(νu + B(u)) , (t, y) ∈[ 0, T] ×[ 0, ∞) and φ(t, x(t), M(t)) = E[ M(T)2\|Ft] = E x(0)2 exp max 0≤u≤T(νu + B(u)) 2 Ft = E x(0)2 exp max max 0≤u≤t(νu + B(u)), max t≤u≤T(νu + B(u)) 2 Ft = E x(0)2 exp max max 0≤u≤t(νu + B(u)), (νt + B(t)) + max 0≤u≤T−t(νu + B(u)) 2 Ft = E x(t)2 exp max max 0≤u≤t(νu + B(u)) −(νt + B(t)), max 0≤u≤T−t(νu + B(u)) 2 Ft = E x(t)2 exp max y, max 0≤u≤T−t(νu + B(u)) 2 y = max 0≤u≤t(νu + B(u)) −(νt + B(t)) = x(t)2G2 t, ln M(t) x(t) , (12) where G2(t, y) = E exp max y, max 0≤u≤T−t(νu + B(u)) 2 , (t, y) ∈[ 0, T] ×[ 0, ∞). where the operator L is defined by L f (t, x, M) = ft(t, x, M) + μxfx(t, x, M) + 1 2x2fxx(t, x, M). The value function V(t, x, M) satisfies V(t, λx, λM) = λ2V(t, x, M), Li et al. Financial Innovation (2017) 3:28 Page 6 of 10 because scaling both x(t) and M(t) by the same positive constant at a time t prior to the terminal time T results in the payoff (x(T)−αM(T))2 being scaled by the same constant. In particular, if U(t, ln z) = V(t, 1, z), 0 ≤t ≤T, z ≥1, then we may determine V(t, x, M) as U(t, ln z) = V(t, 1, z), 0 ≤t ≤T, z ≥1, then we may determine V(t, x, M) as V(t, x, M) = x2V t, 1, M x = x2U t, ln M x , 0 ≤t ≤T, 0 < x ≤M. According to Eq. (13) and expressions of G1 and G2, we have According to Eq. (13) and expressions of G1 and G2, we have x, M) = min t≤τ≤T E x(τ)2 −2αx(τ)ψ(τ, x(τ), M(τ)) + α2φ(τ, x(τ), M(τ)) Ft = min t≤τ≤TE x(τ)2 −2αx(τ)2G1 τ, ln M(τ) x(τ) + α2x(τ)2G2 τ, ln M(τ) x(τ) Ft = min t≤τ≤TE x(τ)2 1 −2αG1 τ, ln M(τ) x(τ) + α2G2 τ, ln M(τ) x(τ) Ft = min t≤τ≤TE x(τ)2G τ, ln M(τ) x(τ) Ft , t≤τ≤T (15) (15) where G(t, y) = 1 −2αG1(t, y) + α2G2(t, y). where G(t, y) = 1 −2αG1(t, y) + α2G2(t, y). Equation (15) implies that Eq. (9) is equivalent to a standard optimal stopping problem with a terminal payoff G and an underlying (adapted) state process Y(t) = ln M(t) x(t) , Y(0) = 0. Y(t) = ln M(t) x(t) , Y(0) = 0. Following the dynamic programming approach we consider the problem below ollowing the dynamic programming approach we consider the problem below U(t, y) = inf τ∈TT−t Et,y[ G(t + τ, Y(t + τ))] , U(t, y) = inf τ∈TT−t Et,y[ G(t + τ, Y(t + τ))] , where Y(t) = y under the probability Pt,x with (t, y) ∈[ 0, T] ×[ 0, ∞) given and fixed, and Ts in general denotes the set of all F-stopping times τ ∈[ 0, s] for s > 0. Theorem 1 The holding region is C = {(t, y) ∈[ 0, T] ×[ 0, ∞) : U(t, y) < G(t, y)}, while the exit region is C = {(t, y) ∈[ 0, T] ×[ 0, ∞) : U(t, y) < G(t, y)}, while the exit region is D = {(t, y) ∈[ 0, T] ×[ 0, ∞) : U(t, y) = G(t, y)}. Also, an optimal exit time is τ ∗= inf t ∈[ 0, T] : t, ln M(t) x(t) ∈D ! . τ ∗= inf t ∈[ 0, T] : t, ln M(t) x(t) ∈D ! . where the operator L is defined by where Y(t) = y under the probability Pt,x with (t, y) ∈[ 0, T] ×[ 0, ∞) given and fixed, and Ts in general denotes the set of all F-stopping times τ ∈[ 0, s] for s > 0. In fact, U satisfies the following dynamic programming equation (or variational inequalities) ⎧ ⎪⎨ ⎪⎩ max { L U, U −G} = 0, (t, y) ∈[ 0, T] ×[ 0, ∞), subject to Uy(t, 0+) = 0, t ∈[ 0, T), U(T, y) = G(T, y), y ∈(0, ∞), (16) (16) re the operator L is defined by where the operator L is defined by where the operator L is defined by L f (t, y) = ft(t, y) −(ν + 2)fy(t, y) + 1 2fyy(t, y) + 2(ν + 1)f (t, y). Hence, the original problem is transferred into finding U. Since x(·) has stationary independent increments and Y(·) is a Markovian process, we rewrite U(t, y) = inf 0≤τ≤T−t E[ G(t + τ, Y y(τ))] , U(t, y) = inf 0≤τ≤T−t E[ G(t + τ, Y y(τ))] , U(t, y) = inf 0≤τ≤T−t E[ G(t + τ, Y y(τ))] , U(t, y) = inf 0≤τ≤T−t E[ G(t + τ, Y y(τ))] , where Y(·) under P is explicitly given as where Y(·) under P is explicitly given as Y y(t) = y ∨ln M(t) x(t) , t ≥0. Y y(t) = y ∨ln M(t) x(t) , t ≥0. Theoretically, we have derived a region in which the venture capitalist may sell the shares they hold, given the pre-determined relationship between her target return and the expected maximum return. Page 7 of 10 Li et al. Financial Innovation (2017) 3:28 Theorem 1 The holding region is Conclusion This paper considers an optimal stopping time point for the investor who is expected to maximize her personal utilities and to minimize the difference between the realized return at the stopping point and her potentially maximum return. Our utility function of a quadratic form is more general than that of a fraction form where the denominator may be zero in Shiryaev et al. (2008). Furthermore, our investment problem, which includes the portfolio in the drift and volatility terms of the dynamic systems, makes the problem including multi-dimensional financial assets more realistic and meaningful. Results and discussion To investigate comparative statics, we present one numerical example in which we change the value of the parameter α. Following the standard approach for estimating the above problem via the finite difference approach, we solve the mathematical formulation given in Eq. (16) by imposing a uniform grid on the (t, y) domain. A Crank-Nicolson scheme is adopted for the discretization of the partial differential equation and the semi-infinite interval for y is truncated at a sufficiently large value of y. The derivative boundary condi- tion is discretized using a forward difference approximation. For the results shown below, we take the grid spacing to be 0.005 for y and 0.001 for t dimensions. Let m = 3. The interest rate of the bond and the appreciation rate of the m stocks are r = 0.05 and (b1, b2, b3)′ = (0.1, 0.12, 0.15)′, respectively, and the volatility matrix is σ = ⎡ ⎢⎣ 0.3000 0 0 0.2000 0.3464 0 0.2500 0.1443 0.4082 ⎤ ⎥⎦. Then Then θ := σ −1(b1 −r, b2 −r, b3 −r)′ = (0.1667, 0.1058, 0.1055)′. Using Theorem 1 and the parameter value of α ranging between 0.8 and 1, we observe that the exit region decreases as the value of α increases, as shown by the combined picture at the right-bottom corner of Fig. 1. Fig. 1 α = 0.8, 0.85, 0.9, 0.95, 1 Page 8 of 10 Li et al. Financial Innovation (2017) 3:28 Appendix A: expression of function G1 We now derive the explicit expression of the function G1, defined by We now derive the explicit expression of the f G1(t, y) = E exp max y, max 0≤u≤T−t(νu + B(u)) = ( ∞ y ezdP max 0≤u≤T−t(νu + B(u)) ≤z + eyP max 0≤u≤T−t(νu + B(u)) ≤y . Note that Note that P max 0≤u≤T−t(νu + B(u)) ≤z = z−ν(T−t) √ T−t −e2νz −z−ν(T−t) √ T−t . According to the standard normal distribution, we have ( ∞ y ezd z−ν(T−t) √ T−t = ( ∞ y ez 1 √2π(T−t)e−(z−ν(T−t))2 2(T−t) dz = e(ν+ 1 2 )(T−t) 1 − y−(ν+1)(T−t) √ T−t . Assume that ν ̸= −1 2. Then Assume that ν ̸= −1 2. Then ( ∞ y ezd e2νz −z−ν(T−t) √ T−t = ( ∞ y 2νe(1+2ν)z −z−ν(T−t) √ T−t dz + ( ∞ y e(1+2ν)zd −z−ν(T−t) √ T−t = − 2ν 1+2ν e(1+2ν)y −y−ν(T−t) √ T−t − 1 1+2ν e ν+ 1 2 (T−t) 1 − y−(ν+1)(T−t) √ T−t . ( ∞ y ezd e2νz −z−ν(T−t) √ T−t = ( ∞ y 2νe(1+2ν)z −z−ν(T−t) √ T−t dz + ( ∞ y e(1+2ν)zd −z−ν(T−t) √ T−t = − 2ν 1+2ν e(1+2ν)y −y−ν(T−t) √ T−t − 1 1+2ν e ν+ 1 2 (T−t) 1 − y−(ν+1)(T−t) √ T−t . Thus G1(t, y) = ey y−ν(T−t) √ T−t − 1 1+2ν e(1+2ν)y −y−ν(T−t) √ T−t + 2(1+ν) 1+2ν e ν+ 1 2 (T−t) 1 − y−(ν+1)(T−t) √ T−t . n addition, note that when ν = −1 2, ( ∞ y ezd e2νz −z−ν(T−t) √ T−t = ( ∞ y ezd e−z −z−ν(T−t) √ T−t = − ( ∞ y −z−ν(T−t) √ T−t dz + ( ∞ y d −z−ν(T−t) √ T−t = y −y−ν(T−t) √ T−t − √ T−t √ 2π e−(x+ν(T−t))2 2(T−t) + ν(T −t) 1 − y+ν(T−t) √ T−t − −y−ν(T−t) √ T−t . Appendix A: expression of function G1 G1(t, y) = ey y−ν(T−t) √ T−t − 1 1+2ν e(1+2ν)y −y−ν(T−t) √ T−t + 2(1+ν) 1+2ν e ν+ 1 2 (T−t) 1 − y−(ν+1)(T−t) √ T−t . n addition, note that when ν = −1 2, ( ∞ y ezd e2νz −z−ν(T−t) √ T−t = ( ∞ y ezd e−z −z−ν(T−t) √ T−t = − ( ∞ y −z−ν(T−t) √ T−t dz + ( ∞ y d −z−ν(T−t) √ T−t = y −y−ν(T−t) √ T−t − √ T−t √ 2π e−(x+ν(T−t))2 2(T−t) + ν(T −t) 1 − y+ν(T−t) √ T−t − −y−ν(T−t) √ T−t . G1(t, y) = ey y−ν(T−t) √ T−t − 1 1+2ν e(1+2ν)y −y−ν(T−t) √ T−t + 2(1+ν) 1+2ν e ν+ 1 2 (T−t) 1 − y−(ν+1)(T−t) √ T−t . In addition, note that when ν = −1 2, Page 9 of 10 Li et al. Financial Innovation (2017) 3:28 Thus Thus G1(t, y) = 1 − y−(ν+1)(T−t) √ T−t −y −y−ν(T−t) √ T−t + √ T−t √ 2π e−(y+ν(T−t))2 2(T−t) −ν(T −t) 1 − y+ν(T−t) √ T−t + ey y−ν(T−t) √ T−t . Appendix B: expression of function G2 Appendix B: expression of function G2 We now derive the explicit expression of the function G2, defined by We now derive the explicit expression of the function G2, defined by G2(t, y) = E exp max y, max 0≤u≤T−t(νu + B(u)) 2 = ( ∞ y e2zdP max 0≤u≤T−t(νu + B(u)) ≤z +e2yP max 0≤u≤T−t(νu + B(u)) ≤y . Note that P max 0≤u≤T−t(νu + B(u)) ≤z = z−ν(T−t) √ T−t −e2νz −z−ν(T−t) √ T−t . According to the standard normal distribution, we have ( ∞ y e2zd z−ν(T−t) √ T−t = ( ∞ y e2z 1 √2π(T−t)e−(z−ν(T−t))2 2(T−t) dz = e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . = e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . Assume that ν ̸= −1. Then Assume that ν ̸= −1. Then Assume that ν ̸= −1. Then ( ∞ y e2zd e2νz −z−ν(T−t) √ T−t = ( ∞ y 2νe2(1+ν)z −z−ν(T−t) √ T−t dz + ( ∞ x e2(1+ν)zd −z−ν(T−t) √ T−t = − ν 1+ν e2(1+ν)y −y−ν(T−t) √ T−t − 1 1+ν e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . Assume that ν ̸= 1. Then ( ∞ y e2zd e2νz −z−ν(T−t) √ T−t = ( ∞ y 2νe2(1+ν)z −z−ν(T−t) √ T−t dz + ( ∞ x e2(1+ν)zd −z−ν(T−t) √ T−t = − ν 1+ν e2(1+ν)y −y−ν(T−t) √ T−t − 1 1+ν e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . Thus G2(t, y) = e2y y−ν(T−t) √ T−t − 1 1+2ν e2(1+ν)y −y−ν(T−t) √ T−t + 2+ν 1+ν e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . Also, note that when ν = −1, ( ∞e2zd e2νz −z−ν(T−t) ( ∞ y e2zd e2νz −z−ν(T−t) √ T−t = ( ∞ y 2νe2(1+ν)z −z−ν(T−t) √ T−t dz + ( ∞ x e2(1+ν)zd −z−ν(T−t) √ T−t = − ν 1+ν e2(1+ν)y −y−ν(T−t) √ T−t − 1 1+ν e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . = − ν 1+ν e2(1+ν)y −y−ν(T−t) √ T−t − 1 1+ν e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . Competing interests Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Appendix B: expression of function G2 Thus Thus G2(t, y) = e2y y−ν(T−t) √ T−t − 1 1+2ν e2(1+ν)y −y−ν(T−t) √ T−t + 2+ν 1+ν e2(ν+1)(T−t) 1 − y−(ν+2)(T−t) √ T−t . √ Also, note that when ν = −1, ( ∞ y e2zd e2νz −z−ν(T−t) √ T−t = ( ∞ y e2zd e−2z −z−ν(T−t) √ T−t = −2 ( ∞ y −z−ν(T−t) √ T−t dz + ( ∞ y d −z−ν(T−t) √ T−t = 2y −y−ν(T−t) √ T−t −2 √ T−t √ 2π e−(y+ν(T−t))2 2(T−t) + 2ν(T −t) 1 − y+ν(T−t) √ T−t − −y−ν(T−t) √ T−t . Also, note that when ν = −1, Also, note that when ν = −1, Page 10 of 10 Li et al. Financial Innovation (2017) 3:28 Thus Thus G2(t, y) = 1 − y−(ν+2)(T−t) √ T−t −2y −y−ν(T−t) √ T−t + 2 √ T−t √ 2π e−(y+ν(T−t))2 2(T−t) −2ν(T −t) 1 − y+ν(T−t) √ T−t + e2y y−ν(T−t) √ T−t . Author details 1 1Department of Applied Mathematics, The Hong Kong Polytechnic University, Hong Kong, China. 2School of Mathematical Sciences, South China Normal University, Guangzhou, China. Received: 8 October 2017 Accepted: 2 November 2017 Funding Funding This work is supported by Research Grants Council of Hong Kong under grant no. 519913 and 15224215, and National Natural Science Foundation of China (No.11571124). Authors’ contributions All authors read and approved the final manuscript. Authors’ contributions Authors contributions All authors read and approved the final manuscript. All authors read and approved the final manuscript. References References Ceci C, Bassan B (2004) Mixed optimal stopping and stochastic control problems with semicontinuous final reward for diffusion processes. Stochast Stochast Rep 76:323–337 Chang MH, Pang T, Yong J (2009) Optimal stopping problem for stochastic differential equations with random coefficients. SIAM J Control Optim 48:941–971 Choi K, Koo H, Kwak D (2004) Optimal stopping of active portfolio management. Ann Econ Finance 5:93–126 Dayanik S, Karatzas I (2003) On the optimal stopping problem for one-dimensional diffusions. 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https://openalex.org/W2065293282	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0062946&type=printable	English	null	The Antimicrobial Compound Xantholysin Defines a New Group of Pseudomonas Cyclic Lipopeptides	PloS one	2,013	cc-by	15,898	Received February 8, 2013; Accepted March 27, 2013; Published May 17, 2013 Received February 8, 2013; Accepted March 27, 2013; Published May 17, 2013 Copyright: 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the KU Leuven Research Council Fund through a KU Leuven-Zhejiang University interuniversity SBA fellowship to WL and grant GOA/011/2008 to RDM. The Fund for Scientific Research – Flanders (FWO-Vlaanderen) is gratefully acknowledged for a PhD fellowship and a postdoctoral mandate to MDV and DS, respectively, and for joint research projects to AM and JCM (G.0901.10 and G.0422.13). Funding for mass spectrometry was obtained from the Hercules Foundation of the Flemish Government (grant 20100225-7). The 700 MHz and 500 MHz NMR equipment are part of the Interuniversity NMR Facility and are funded by the Flemish Government (FFEU-ZWAP) and the Hercules Foundation, respectively. 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: rene.demot@biw.kuleuven.be Wen Li1, Hassan Rokni-Zadeh1, Matthias De Vleeschouwer2,3, Maarten G. K. Ghequire1, Davy Sinnaeve2, Guan-Lin Xie4, Jef Rozenski5, Annemieke Madder3, Jose´ C. Martins2, Rene´ De Mot1* Wen Li1, Hassan Rokni-Zadeh1, Matthias De Vleeschouwer2,3, Maarten G. K. Ghequire1, Davy Sinnaeve2, Guan-Lin Xie4, Jef Rozenski5, Annemieke Madder3, Jose´ C. Martins2, Rene´ De Mot1* 1 Centre of Microbial and Plant Genetics, Department of Microbial and Molecular Systems, University of Leuven, Heverlee-Leuven, Belgium, 2 NMR and Structure Analysis Unit, Department of Organic Chemistry, Ghent University, Gent, Belgium, 3 Organic and Biomimetic Chemistry Research Group, Department of Organic Chemistry, Ghent University, Gent, Belgium, 4 State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China, 5 Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium Citation: Li W, Rokni-Zadeh H, De Vleeschouwer M, Ghequire MGK, Sinnaeve D, et al. (2013) The Antimicrobial Compound Xantholysin Defines a New Group of Pseudomonas Cyclic Lipopeptides. PLoS ONE 8(5): e62946. doi:10.1371/journal.pone.0062946 Abstract The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptide’s capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin- producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysin’s macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3- hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cyclohepta- peptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides. Citation: Li W, Rokni-Zadeh H, De Vleeschouwer M, Ghequire MGK, Sinnaeve D, et al. (2013) The Antimicrobial Compound Xantholysin Defines a New Group of Pseudomonas Cyclic Lipopeptides. PLoS ONE 8(5): e62946. doi:10.1371/journal.pone.0062946 Editor: Yung Fu Chang Cornell University United States of America Editor: Yung-Fu Chang, Cornell University, United States of America Identification of P. putida BW11M1 Mutants Lacking Anti- Xanthomonas Activity y To identify mutants lacking anti-Xanthomonas activity, a P. putida BW11M1 plasposon mutant library [35] was screened using X. alfalfae subsp. alfalfae LMG 497 as indicator strain. Mutants lacking anti-LMG 497 activity were isolated and subjected to plasposon rescue [36] for subsequent sequencing of plasposon-flanking DNA (Table S1). The majority of the mutants (23 out of 34) was interrupted in genes showing similarity to NRPS genes, in particular to those of Pseudomonas entomophila L48 encoding the enzymes EtlA, EtlB and EtlC that synthesize the cyclic lipopeptide entolysin [10]. In mutant CMPG2201, a BW11M1 gene was hit that showed homology to etlR, the etlA-linked regulatory gene required for entolysin production [10]. In addition to these clustered insertions pointing to biosynthesis of a putative (lipo)peptide, additional mutations in unlinked genes were identified (Table S1). Although to be confirmed by phenotypic complementation with the respective wild-type genes, these mutations suggest that modified cell envelope characteristics (lipopolysaccharide, exopolysaccharide, large surface protein) may impair secretion of the secondary metabolite and that its production may be triggered by stress factors (recB and clpB mutants). The latter would be in line with the involvement of DnaK and ClpP in the production of the lipopeptides putisolvin by P. putida [37] and massetolide by P. fluorescens [15], respectively. Several Pseudomonas lipopeptides are harmful to fungi and oomycetes but their antibacterial activities are mostly confined to Gram-positive bacteria, while in general Gram-negative bacteria seem to be better protected by their different cell envelope architecture with a surface-exposed outer membrane [24]. It was noted, however, that a number of Xanthomonas species are susceptible to the cyclic lipopeptide WLIP, a member of the viscosin group, produced by Pseudomonas putida RW10S2 [17], Pseudomonas fluorescens LMG 5329 [18], and Pseudomonas aurantiaca PB-St2 [25]. The c-proteobacterial genus Xanthomonas represents a major group of plant-associated bacteria, most of which cause plant diseases of economically important mono- and dicot crops [26,27]. In a recent survey, three of its species were ranked among the top ten pathogenic bacteria of scientific and economic interest [28]. In plant environments, competition for nutrients and space are likely to occur between Xanthomonas and Pseudomonas as metagenomic surveys have revealed that both genera of c-proteobacteria constitute an important fraction of the microbial communities in the rhizosphere and phyllosphere of diverse plants [29–32]. Xanthomonad-inhibitory Activity of P. putida BW11M1 Xanthomonad-inhibitory Activity of P. putida BW11M1 Xanthomonad-inhibitory Activity of P. putida BW11M1 Screening of a collection of Pseudomonas strains isolated from tropical crop roots [33] revealed a broad xanthomonad-inhibitory activity for the banana rhizosphere isolate P. putida BW11M1. Using an agar diffusion assay, spotted BW11M1 cells produced a growth inhibition halo with pathovars of different Xanthomonas species as indicator overlay (Fig. 1). The growth inhibitory pattern for some of these strains, such as X. translucens pv. cerealis LMG 679 and X. vasicola pv. musacearum LMG 785, consists of a clear halo surrounded by a larger somewhat turbid halo, suggesting sensitivity to more than one BW11M1 metabolite. As strain BW11M1 is capable of killing pseudomonads by production of the lectin-like toxin LlpA [34], it was verified whether this bacteriocin might also be responsible for the observed growth inhibition of xanthomonads, phylogenetic relatives of pseudomonads. Howev- er, the latter antagonism was not affected in a BW11M1 llpA mutant [35] and an Escherichia coli strain producing recombinant LlpA [35] displayed no activity against Xanthomonas species, demonstrating LlpA not to be involved (data not shown). Furthermore, halo formation was not abolished by a non-specific protease spotted close to BW11M1 producer cells, pointing to a non-proteinaceous nature of the molecule(s) causing halo forma- tion in the lawn of Xanthomonas cells (data not shown). The Pseudomonas biosynthetic genes for syringomycin, syringo- peptin, syringafactin, arthrofactin, viscosin, orfamide, massetolide, putisolvin, entolysin, WLIP, and cichofactin have been described [5,8–18]. The encoded nonribosomal peptide synthetases (NRPSs) are composed of multiple modules, each consisting of an adenylation (A) domain responsible for amino acid selection and activation, a thiolation (T) domain responsible for thioesterifica- tion of the activated substrate, and a condensation (C) domain that catalyzes peptide bond formation between two amino acids. The starter condensation domain in the initiating enzyme (C1) catalyzes acylation of the first amino acid, thereby linking the lipid moiety to the oligopeptide [19,20]. Most known Pseudomonas lipopeptide systems are of Type A, obeying the ‘‘co-linearity rule’’. This indicates that the order and number of the NRPS modules are co-linear to the amino acid sequence of the peptide product [5,21]. Another characteristic is the frequent occurrence of a tandem thioesterase (TE) domain in the terminating NRPS domain required for release, usually with concomitant cyclization, of the mature peptide product [6]. Xanthomonad-inhibitory Activity of P. putida BW11M1 Multiple D-configured amino acids have been identified in Pseudomonas lipopeptides with known stereochemistry, but the corresponding synthetases lack standalone epimerization (E) domains, as found in for instance Bacillus lipopeptide NRPSs [6] or Pseudomonas pyoverdine synthetases [22]. This is attributed to the activity of dual C/E domains with embedded L-to-D epimerization capacity acting on an amino acid loaded on the T domain of a previous module [23]. Introduction pyoluteorin, or lipopeptides [2]. Together with Bacillus, Pseudomonas are prominent producers of lipopeptides with a range of different biological activities: antagonism of microbial competitors, protec- tion against predators, facilitation of surface motility, biofilm formation, contribution to virulence of plant pathogens, triggering of the defense response in plants [3]. Certain genera of soil-dwelling and plant-associated bacteria, such as Streptomycetes, Myxobacteria, Bacilli, Pseudomonads, and Burkholderia, display a highly versatile secondary metabolism and, hence, have proven valuable sources for structurally very diverse metabolites with useful biological activities, including antibiotics. Whereas Streptomyces and other actinomycetes are major producers of clinical antibiotics, several antimicrobials from other soil bacteria, such as Bacillus and Pseudomonas, contribute to their capacity to suppress fungal plant diseases [1]. Such biocontrol effect on phytopathogenic fungi by Pseudomonas strains has been ascribed to in situ production of structurally diverse molecules: 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, Based on similarities in peptide length (ranging from 8 to 25 residues) and amino acid sequence, many of the lipopeptides produced by Pseudomonas strains can be assigned to specific groups, each named after a prototype compound: viscosin, amphisin, syringomycin, syringopeptin, or tolaasin [4–6]. The largest group with six subtypes is represented by viscosin and its analogs massetolide, viscosinamide, pseudodesmin, pseudophomin, and May 2013 \| Volume 8 \| Issue 5 \| e62946 May 2013 \| Volume 8 \| Issue 5 \| e62946 1 PLOS ONE \| www.plosone.org Antimicrobial Pseudomonas putida Lipopeptide Results and Discussion WLIP (white line-inducing principle). In addition, several ‘orphan’ lipopeptides not belonging to one of the known families have been described, namely putisolvin [7], orfamide [8], syringafactin [9], and entolysin [10]. Quite often a particular Pseudomonas strains can produce variants of a particular lipopeptide by attaching a different fatty acid or by incorporation of a similar amino acid at a certain position due to relaxed substrate specificity of the biosynthetic enzymes. With the exception of syringafactins and its analogs, cichofactins [11], all of these molecules are cyclic lipopeptides, as they contain a macrocyclic lactone ring of variable size formed between the carboxyterminal residue of the peptide and an internal amino acid (serine or threonine). Identification of P. putida BW11M1 Mutants Lacking Anti- Xanthomonas Activity This report describes the characterization of Xanthomonas-antagonistic activity of Pseudomonas putida BW11M1, a strain isolated from banana rhizosphere in Sri Lanka, revealing the involvement of a new type of lipopeptide. Further analysis focused on the NRPS-related genes of P. putida BW11M1 apparently involved in biosynthesis of the substance inhibitory to Xanthomonas, tentatively designated xantholysin. In view of the anticipated large genomic DNA regions encoding multi-modular megasynthases, a fosmid library of genomic BW11M1 fragments was constructed. NRPS-specific PCR primers were designed based on the plasposon-flanking DNA sequences for the NRPS mutants listed in Table S1. Three amplicon-positive fosmids, pCMPG6126, pCMPG6127 and pCMPG6128 with estimated insert sizes of about 38 kb, 41 kb, and 40 kb respec- tively, were selected. Sequence analysis of a shotgun library constructed for the three pooled fosmids yielded six insert contigs that enabled the localization of three NRPS genes, designated xtlA, PLOS ONE \| www.plosone.org May 2013 \| Volume 8 \| Issue 5 \| e62946 2 Antimicrobial Pseudomonas putida Lipopeptide Figure 1. Growth inhibition of Xanthomonas species by P. putida BW11M1. Spotted BW11M1 cells (2 ml of 107 CFU/ml) were overlayed with Xanthomonas indicator cells. (A) X. alfalfae subsp. alfalfae LMG 497; (B) X. axonopodis pv. manihotis LMG 784; (C) X. hortorum pv. hederae LMG 7411; (D) X. sacchari LMG 471; (E) X. translucens pv. cerealis LMG 679; (F) X. translucens pv. graminis LMG 726; (G) X. translucens pv. hordei LMG 737; (H) X. vasicola pv. holcicola LMG 736; (I) X. vasicola pv. musacearum LMG 785. doi:10.1371/journal.pone.0062946.g001 Figure 1. Growth inhibition of Xanthomonas species by P. putida BW11M1. Spotted BW11M1 cells (2 ml of 107 CFU/ml) were overlayed with Xanthomonas indicator cells. (A) X. alfalfae subsp. alfalfae LMG 497; (B) X. axonopodis pv. manihotis LMG 784; (C) X. hortorum pv. hederae LMG 7411; (D) X. sacchari LMG 471; (E) X. translucens pv. cerealis LMG 679; (F) X. translucens pv. graminis LMG 726; (G) X. translucens pv. hordei LMG 737; (H) X. vasicola pv. holcicola LMG 736; (I) X. vasicola pv. musacearum LMG 785. doi:10.1371/journal.pone.0062946.g001 this approach revealed that the XtlA-C1 domain clusters with known starter domains in Pseudomonas lipopeptide synthetases (Fig. S1), suggesting it to attach a fatty acid to the first amino acid. Only two domains (XtlC-C12, XtlC-C13) are predicted to be conven- tional C domains, the remaining eleven C domains being assigned to the C/E type (Fig. 2). Features of the P. putida BW11M1 NRPS System Required for Xanthomonas Inhibition The termination module of XtlC harbors two thioesterase domains (TE1/TE2 tandem) which is similar to most known Pseudomonas lipopeptide biosynthesis systems, except for the syringomycin group [6,43,44] (Fig. 2). Such thioesterase tandem also occurs in LybB from the c-proteobacterium Lysobacter sp. ATCC 53042 that catalyzes release and concomitant macro- cyclization of the peptide lysobactin by its TE1 domain, while TE2 serves to deacylate misprimed T domains [42]. Analysis of thioesterase domains from several Pseudomonas strains (Fig. S2) shows that XtlC TE1 and TE2 domains indeed form separate clusters and the pairwise similarities with the LybB domains suggest a possible thioesterase task division between product formation (TE1) and repair (TE2). The better sequence conser- vation among TE2 domains compared to the TE1 sequences (visualized by tighter TE2 clustering in Fig. S2) favors such general editing function. Possibly, the TE1 catalytic activity for depsi-bond formation co-evolved stronger for various peptide substrates in which lactone rings of different sizes are formed between a terminal residue and an internal serine or threonine. The hydroxylated side-chain of the sole serine residue predicted for xantholysin represents a likely candidate site for cyclization. In silico analysis revealed that the three NRPSs from strain BW11M1, designated XtlA, XtlB and XtlC, comprise two, eight and four modules, respectively, each composed of a condensation (C), adenylation (A) and thiolation (T) domain (Fig. 2). A thioesterase (TE) domain tandem is located at the carboxyterminal end of XtlC. This architecture is quite similar to the P. entomophila biosynthetic system EtlA-EtlB-EtlC assembling the cyclic lipote- tradecapeptide entolysin [10]. The level of pairwise amino acid sequence identities for XtlA/EtlA (75%), XtlB/EtlB (69%), and XtlC/EtlC (72%) further suggests that a different lipopeptide is synthesized by the sequential actions of the initiatory enzyme XtlA, XtlB and the terminating enzyme XtlC. From comparison of empirical data on the adenylating activities of NRPS enzymes it has become possible to make enzyme primary sequence-based predictions about the kind of substrate that is loaded onto a specific module. The NPRSpredictor2 tool reports the probable A-domain selectivity by comparative analysis of a 10- amino acid diagnostic motif contained within the 34-amino acid active site signature sequence ([38]; Table S2). An alternative approach consists of a phylogenetic comparison of novel A domains with those of functionally characterized NRPS systems, which was carried out here for the known Pseudomonas lipopeptide systems (Fig. 3). Features of the P. putida BW11M1 NRPS System Required for Xanthomonas Inhibition Application of both approaches suggested the following sequence for the peptide moiety in xantholysin: Leu- Glu/Asp-Gln-Val-Leu/Ile-Gln-Ser-Val-Leu/Ile-Gln-Leu-Leu- Gln-Ile/Val/Leu. The Asp residue at the second position, suggested by NRPSpredictor2 analysis (Table S2), deviates from the phylogeny-based inference (Glu). The peptide sequence of entolysin A differs from the xantholysin prediction in at least six positions (underlined): Leu-Glu-Gln-Val-Leu-Gln-Val-Leu-Gln- Ser-Val-Leu-Ser-Ile [10,39]). Identification of P. putida BW11M1 Mutants Lacking Anti- Xanthomonas Activity Such quantitative predominance of C/E domains over regular C domains is a prominent feature of Pseudomonas lipopeptide biosynthetic systems (Fig. S1), which typically lack standalone epimerizing domains. In principal, a C/E domain epimerizes the configuration of the amino acid that is loaded onto the T domain of the previous module [23,42]. This analysis suggests that most of the xantholysin residues would be in the D configuration. However, a certain number of deviations from such sequence-based prediction of Ca-epimerization have been noted among Pseudomonas lipopeptides [8,15,17] and the predicted stereochemistry requires experimental validation. xtlB and xtlC (acronym xtl referring to xanthomonad-lytic activity), in two unlinked DNA regions (Fig. 2). All the plasposon insertions in the NRPS mutants are confined to these three genes. In addition, a putative regulatory gene interrupted in mutant CMPG2201, designated xtlR, is located upstream and oriented divergently of xtlA. Several Pseudomonas strains contain similarly organized three-membered NRPS systems consisting of an initiatory NRPS gene linked with a cognate LuxR-family gene, but located remotely from the operon with the two other NRPS genes. This genetic backbone structure was described for biosynthesis of viscosin [14], massetolide [15], entolysin [10], and WLIP [17,18]. This pronounced analogy suggested that a non-ribosomal peptide is the likely product of the xtl-encoded NRPSs. Xantholysin Production is Dependent on a Cognate LuxR-family Regulator 3) and assuming consecutive co-linear biosynthesis by XtlA, XtlB and XtlC. The experimentally determined structure of the main biosynthetic product, xantholysin A, is shown. doi:10.1371/journal.pone.0062946.g002 The XtlD-XtlE-XtlF system and its counterparts in other lipopeptide-producing pseudomonads are similar to the ATP- dependent tripartite macrolide efflux transporter MacA-MacB- TolC system of E. coli [49], with the OprM-like outer membrane proteins such as XtlF presumably operating as TolC equivalents in these pseudomonads [50]. These putative BW11M1 transporter components also showed the highest similarity to those of the entolysin system (Fig. S3.B). Inactivation of the Mac-like Pseudomonas genes downstream of the putisolvin [16], arthrofactin [47], and syringopeptin [51] biosynthetic operons cause strongly reduced but not completely abolished lipopeptide production, indicating a major but non-exclusive role in export. Such redundancy in lipopeptide export capacity may explain why no xantholysin null mutants with insertions in these putative transporter genes were identified. The involvement of an OprM homologue in lipopeptide production by Pseudomonas has not yet been experimentally verified. With its closest homologue, EtlR of P. entomophila required for entolysin production [10], XtlR shares 75% amino acid identity. To confirm the involvement of XtlR in regulation of xantholysin production, mutant CMPG2201 was transformed with pCMPG6204, carrying a cloned wild-type copy of xtlR. The complemented mutant CMPG2201 showed anti-Xanthomonas activity but somewhat lower than wild-type level (Fig. 4A). A regulatory role for the equivalent genes was also reported for production of syringafactin (syfR; [9]), putisolvin (psoR; [16]), viscosin (viscAR; [45]), arthrofactin (arfF; [46]), and WLIP (wlpR; [17]). A downstream-positioned LuxR-type regulator gene (viscBCR; [45]) is also involved in viscosin production, but its counterpart (pspto2833) is not required for production of syringafactin [9]. No additional gene similar to viscBCR is present downstream of the xtlBC operon (data not shown), which is also the case for the wlpBC (WLIP) and etlBC (entolysin) operons [10,17]. The XtlD-XtlE-XtlF system and its counterparts in other lipopeptide-producing pseudomonads are similar to the ATP- dependent tripartite macrolide efflux transporter MacA-MacB- TolC system of E. coli [49], with the OprM-like outer membrane proteins such as XtlF presumably operating as TolC equivalents in these pseudomonads [50]. These putative BW11M1 transporter components also showed the highest similarity to those of the entolysin system (Fig. S3.B). Xantholysin Production is Dependent on a Cognate LuxR-family Regulator Inactivation of the Mac-like Pseudomonas genes downstream of the putisolvin [16], arthrofactin [47], and syringopeptin [51] biosynthetic operons cause strongly reduced but not completely abolished lipopeptide production, indicating a major but non-exclusive role in export. Such redundancy in lipopeptide export capacity may explain why no xantholysin null mutants with insertions in these putative transporter genes were identified. The involvement of an OprM homologue in lipopeptide production by Pseudomonas has not yet been experimentally verified. Transporter Genes are Clustered with the Xantholysin Biosynthetic Genes The Antibacterial Activity of Xantholysin is not Restricted to Xanthomonads Xantholysin Production is Dependent on a Cognate LuxR-family Regulator The divergently transcribed gene upstream of xtlA, inactivated in mutant CMPG2201 is predicted to encode a LuxR-family protein (Fig. 2). Genes encoding such LuxR-type regulators have been identified near the coding regions of most Pseudomonas lipopeptide-synthesizing NRPS genes [45]. These proteins, specif- ically controlling lipopeptide production, constitute a subfamily of regulators that lack a N-acylhomoserine lactone-binding domain and, consequently, are not involved in quorum sensing. The putative regulator of P. putida BW11M1, tentatively designated XtlR, clearly belongs to this sub-group of regulators (Fig. S3.A). Different types of C domains can be discerned by sequence- based phylogenetic analysis [10,15,17,18,40,41]. Application of May 2013 \| Volume 8 \| Issue 5 \| e62946 3 PLOS ONE \| www.plosone.org 3 Antimicrobial Pseudomonas putida Lipopeptide Figure 2. Xantholysin biosynthetic gene clusters of P. putida BW11M1. The organization of the genomic regions with the xantholysin synthetases genes (xtlA, xtlB and xtlC), the associated regulatory gene (xtlR) and putative export genes (xtlD, xtlE and xtlF) is shown (GenBank accession numbers: KC297505 (xtlFRA); KC297506 (xtlBCDE), together with the position of the sequenced fosmid-cloned genomic fragments. Plasposon insertion sites generating mutants without antagonistic activity X. alfalfae subsp. alfalfae LMG 497 are indicated (solid lines). Mutants selected for further phenotypic characterization are highlighted in blue font. For the encoded NRPS enzymes, the modular composition and domain architecture is visualized. The predicted amino acid sequence is based on the substrate specificity of the 14 modules, as inferred by phylogenetic analysis of Pseudomonas A-domain sequences (Fig. 3) and assuming consecutive co-linear biosynthesis by XtlA, XtlB and XtlC. The experimentally determined structure of the main biosynthetic product, xantholysin A, is shown. doi:10.1371/journal.pone.0062946.g002 Figure 2. Xantholysin biosynthetic gene clusters of P. putida BW11M1. The organization of the genomic regions with the xantholysin synthetases genes (xtlA, xtlB and xtlC), the associated regulatory gene (xtlR) and putative export genes (xtlD, xtlE and xtlF) is shown (GenBank accession numbers: KC297505 (xtlFRA); KC297506 (xtlBCDE), together with the position of the sequenced fosmid-cloned genomic fragments. Plasposon insertion sites generating mutants without antagonistic activity X. alfalfae subsp. alfalfae LMG 497 are indicated (solid lines). Mutants selected for further phenotypic characterization are highlighted in blue font. For the encoded NRPS enzymes, the modular composition and domain architecture is visualized. The predicted amino acid sequence is based on the substrate specificity of the 14 modules, as inferred by phylogenetic analysis of Pseudomonas A-domain sequences (Fig. The Antibacterial Activity of Xantholysin is not Restricted to Xanthomonads The genes downstream of xtlC (xtlD-xtlE) and xtlR (xtlF) potentially encode components of an export system for xantholysin (Fig. 2). This genomic organization, with equivalent genes flanking the initiating and terminating NRPS genes, is a recurrent theme for several Pseudomonas lipopeptide gene clusters [10,15–18,47,48]. X. alfalfae subsp. alfalfae LMG 497 was used as an indicator strain to identify BW11M1 mutants lacking xantholysin produc- tion. To assess whether the observed inhibition of other Xanthomonas strains also relies on this lipopeptide system, repre- May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 4 Antimicrobial Pseudomonas putida Lipopeptide sentative anti-LMG 497 null mutants with a disrupted xtlA, xtlB, the BW11M1 spot for sensitive strains such as X. axonopodis pv. Figure 3. Phylogeny-based substrate specificity prediction of xantholysin synthetases. Cladogram of maximum-likelihood tree inferred from amino acid sequence alignment of adenylation (A) domains extracted from functionally characterized Pseudomonas NRPSs. Lipopeptide-specific codes used for NRPS enzymes: Arf (arthrofactin, P. fluorescens MIS38); Etl (entolysin, P. entomophila L48); Mass (massetolide, P. fluorescens SS101); Ofa (orfamide, P. fluorescens Pf-5); Pso (putisolvin, P. putida PCL1445); Syf (syringafactin, P. syringae DC3000); Syp (syringopeptin, P. syringae pv. syringae B301D); Syr (syringomycin, P. syringae pv. syringae strain B301D); Visc (viscosin, P. fluorescens SBW25); Wip (WLIP, P. fluorescens LMG 5329); Wlp (WLIP, P. putida RW10S2); Xtl (xantholysin, P. putida BW11M1; highlighted in larger bold font). For each domain the substrate specificity is indicated in parentheses using the standard amino acid three-letter code (for xantholysin, as determined in this work). Non-protein amino acids are annotated as follows: allo-threonine (aTHR); 2,3-dehydro-2-aminobutyric acid (DHB); 2,4-diaminobutyric acid (DAB); 3-hydroxyaspartate (OH-ASP); unidentified residue (Leu or Ile) in putisolvin II (XLE). Clusters comprising xantholysin domains are highlighted in different colors. The tree was rooted with the divergent SyrB1 domain. doi:10.1371/journal.pone.0062946.g003 Figure 3. Phylogeny-based substrate specificity prediction of xantholysin synthetases. Cladogram of maximum-likelihood tree inferred from amino acid sequence alignment of adenylation (A) domains extracted from functionally characterized Pseudomonas NRPSs. Lipopeptide-specific codes used for NRPS enzymes: Arf (arthrofactin, P. fluorescens MIS38); Etl (entolysin, P. entomophila L48); Mass (massetolide, P. fluorescens SS101); Ofa (orfamide, P. fluorescens Pf-5); Pso (putisolvin, P. putida PCL1445); Syf (syringafactin, P. syringae DC3000); Syp (syringopeptin, P. syringae pv. syringae B301D); Syr (syringomycin, P. syringae pv. syringae strain B301D); Visc (viscosin, P. fluorescens SBW25); Wip (WLIP, P. fluorescens LMG 5329); Wlp (WLIP, P. putida RW10S2); Xtl (xantholysin, P. The Antibacterial Activity of Xantholysin is not Restricted to Xanthomonads putida BW11M1; highlighted in larger bold font). For each domain the substrate specificity is indicated in parentheses using the standard amino acid three-letter code (for xantholysin, as determined in this work). Non-protein amino acids are annotated as follows: allo-threonine (aTHR); 2,3-dehydro-2-aminobutyric acid (DHB); 2,4-diaminobutyric acid (DAB); 3-hydroxyaspartate (OH-ASP); unidentified residue (Leu or Ile) in putisolvin II (XLE). Clusters comprising xantholysin domains are highlighted in different colors. The tree was rooted with the divergent SyrB1 domain. doi:10 1371/journal pone 0062946 g003 the BW11M1 spot for sensitive strains such as X. axonopodis pv. manihotis LMG 784 (Fig. 4A). For strains such as X. translucens pv. cerealis LMG 679 with two concentric growth-inhibitory halo’s, only the smaller clear halo disappeared. The latter was restored sentative anti-LMG 497 null mutants with a disrupted xtlA, xtlB, xtlC or xtlR gene, were assayed for antagonism against other Xanthomonas strains (Table S3). The lack of xantholysin production coincided with disappearance of the single clear halo surrounding May 2013 \| Volume 8 \| Issue 5 \| e62946 May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 5 PLOS ONE \| www.plosone.org Antimicrobial Pseudomonas putida Lipopeptide Figure 4. Phenotypes of P. putida BW11M1 and xantholysin-deficient mutants. (A) Antagonistic activity against X. axonopodis pv. manihotis LMG 784. (B) Antagonistic activity against X. translucens pv. cerealis LMG 679. (C) Formation of brown blotch on sliced Agaricus bisporus tissue. (D) Hemolysis on horse blood TSB agar plate. (E) Swarming on 0.8% TSB agar plate. (F) Biofilm formation on pegs visualized by staining of adherent cells. WT: BW11M1 wild type; xtlA, xtlB, xtlC, and xtlR: mutants CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively; xltR+: mutant CMPG2201 with pCMPG6204 containing xtlR of P. putida BW11M1. The phenotypes shown for the selected xtlA, xtlB and xtlC mutants are representative for the other xtl NRPS mutants (Table S1). The quantitative data for biofilm formation are shown in Fig. 5. doi:10.1371/journal.pone.0062946.g004 Figure 4. Phenotypes of P. putida BW11M1 and xantholysin-deficient mutants. (A) Antagonistic activity against X. axonopodis pv. manihotis LMG 784. (B) Antagonistic activity against X. translucens pv. cerealis LMG 679. (C) Formation of brown blotch on sliced Agaricus bisporus tissue. (D) Hemolysis on horse blood TSB agar plate. (E) Swarming on 0.8% TSB agar plate. (F) Biofilm formation on pegs visualized by staining of adherent cells. Antimicrobial Pseudomonas putida Lipopeptide when assaying the complemented xtlR mutant, also linking this phenotype to xantholysin production (Fig. 4B). With these indicator strains, the larger turbid halo remained visible but the zone of inhibition caused by the BW11M1 mutants was somewhat reduced compared to the wild type (Table S3). The sensitivity of xanthomonads to xantholysin from P. putida BW11M1 shows qualitative and quantitative differences compared to WLIP- mediated inhibition by P. putida RW10S2 [17]. For instance, growth of the strain most sensitive to WLIP, X. citri pv. malvacearum LMG 761, is only weakly affected by xantholysin production (Table S3). when assaying the complemented xtlR mutant, also linking this phenotype to xantholysin production (Fig. 4B). With these indicator strains, the larger turbid halo remained visible but the zone of inhibition caused by the BW11M1 mutants was somewhat reduced compared to the wild type (Table S3). The sensitivity of xanthomonads to xantholysin from P. putida BW11M1 shows qualitative and quantitative differences compared to WLIP- mediated inhibition by P. putida RW10S2 [17]. For instance, growth of the strain most sensitive to WLIP, X. citri pv. malvacearum LMG 761, is only weakly affected by xantholysin production (Table S3). several ascomycetes, the basidiomycete Rhizoctonia solani was also growth-inhibited. P. putida BW11M1 cells applied on the sliced fruiting body of the basidiomycete Agaricus bisporus [52] resulted in discoloration. This tissue damage was not visible for the xantholysin-deficient mutants, indicating it to be dependent on xantholysin production (Fig. 4C). Tissue discoloration was also observed for the complemented xtlR mutant CMPG2201 carrying pCMPG6204. No such blotch-like symptoms were observed following application of a BW11M1 cell suspension onto intact mushroom caps (data not shown). Antifungal activity has been reported for several cyclic lipopeptides belonging to different structural groups [3]. These include tolaasin, a mycotoxin from the mushroom pathogen P. tolaasii, and its analog sclerosin [53–55], amphisin [56], viscosina- mide [57], thanamycin (syringomycin group; [58]), and syringo- peptin [59]. Immobilization and lysis of oomycete zoospores is caused by several viscosin-group members [3]. The in vitro antifungal activity of entolysin has not been investigated, but production of entolysin is not involved in the biocontrol activity of P. entomophila L48 in a Pythium ultimum/cucumber setup [10]. To further examine the antibacterial capacity of strain BW11M1 and the role played by xantholysin production in this, an expanded indicator panel composed of Gram-negative and Gram-positive bacteria (Table S4) was tested against BW11M1 wild type and selected mutants. P. Hemolytic Activity of P. putida BW11M1 is Mediated by Xantholysin Hemolytic activity has been reported for a number of lipopeptides [10,17,48,54]. This phenotype can be readily visualized by formation of a discoloured halo surrounding a colony grown on blood agar. P. putida BW11M1 also shows hemolytic activity, whereas mutants deficient in anti-Xanthomonas activity are devoid of it (Fig. 4D). In the complemented xtlR mutant CMPG2201 carrying pCMPG6204, hemolysis was restored, in parallel with anti-Xanthomonas activity (Fig. 4A, 4B), thus identifying xantholysin as the hemolytic factor produced by strain BW11M1. Hemolysis by the structurally related lipopeptide entolysin does not contribute to virulence of its entomopathogenic producer [10]. Growth of most of the Gram-positive bacteria strains tested (seven out of nine) was inhibited by strain BW11M1: the firmicutes Bacillus megaterium ATCC 13632, Bacillus subtilis LMG 7135 and Staphylococcus aureus ATCC 6358, and the actinomycetes Mycobac- terium smegmatis DSM 43756, Rhodococcus erythropolis N11, Streptomyces coelicolor A3(2) and Streptomyces lividans TK24. The residual antagonistic activity towards these Gram-positive bacteria dis- played by the xantholysin-deficient mutants again points to production of (an)other growth-inhibitory factor(s) by P. putida BW11M1 (Table S3). The antagonistic activity conferred by xantholysin production based on mutant analysis was confirmed for selected strains with purified xantholysin (purification described below; Fig. S4). When testing this sample against rice pathogen X. oryzae pv. oryzae, strains PXO99 and PXO112 proved sensitive, whereas growth of strain PXO340 was not affected (data not shown). Antimicrobial Pseudomonas putida Lipopeptide putida BW11M1 did not display a broad inhibitory activity among other Gram-negative bacteria, with six out of seventeen additional Gram-negative bacteria tested being inhibited by strain BW11M1: the a-proteobacteria Azospir- illum brasilense Sp7 and Sphingomonas wittichii RW1, the b- proteobacteria Burkholderia vietnamiensis LMG 10927 and Variovorax paradoxus LMG 1797, and the c-proteobacteria Aeromonas hydrophila ATCC 7966 and Erwinia amylovora CFBP 1430 (Table S3). For the Aeromonas, Burkholderia and Erwinia strains, xantholysin production appears to be solely responsible for inhibition. The antagonistic effect of strain BW11M1 exerted on pseudomonads, including members of the P. syringae group, was not abolished in the xantholysin mutants (data not shown), confirming that this is bacteriocin-mediated [34]. Xantholysin is Required for Swarming and Contributes to Biofilm Formation For several Pseudomonas strains the involvement of lipopeptides in swarming and biofilm formation has been reported [3,17,18,48]. It was investigated whether these phenotypes are equally associated with xantholysin production. The P. putida BW11M1 mutants impaired in anti-Xanthomonas antagonism were also deficient in swarming ability on soft agar (0.8%), compared to the strong surface translocation capacity of the wild-type strain (Fig. 4E). Swarming of the regulatory mutant (CMPG2201), carrying a cloned xtlR copy (pCMPG6204), was comparable to the wild-type behavior (Fig. 4E). This reveals a primary role for xantholysin in swarming by P. putida BW11M1. Swarming capacity is also affected in P. entomophila mutants lacking entolysin production [10]. The inhibition of Gram-negative bacteria is a property not typically associated with lipopeptides, whereas Gram-positive bacteria are generally more susceptible [3]. In a comparative study of the antagonistic activity of five Pseudomonas lipopeptides (massetolide, syringomycin, orfamide, arthrofactin, and entolysin), no significant inhibition of Gram-negative bacteria was observed [24]. Conversely, among Gram-positive bacteria, an Arthrobacter strain (actinomycete) was susceptible to all tested compounds, except orfamide. Arthrofactin was also active against other actinomycetes (Corynebacterium, Mycobacterium). Compared to wild-type BW11M1 cells, the mutants deficient in anti-Xanthomonas activity showed significantly reduced biofilm formation at 30uC, but still retained almost half of the wild-type capacity (Fig. 4F, Fig. 5). This indicates that xantholysin contributes to BW11M1 biofilm formation under these experi- mental conditions, acting together with other factors. Biofilm formation by the complemented regulatory mutant was increased to about 80% of wild-type biofilm formation (Fig. 5). The Broad Antifungal Activity of P. putida BW11M1 is mainly Dependent on Xantholysin Production The Antibacterial Activity of Xantholysin is not Restricted to Xanthomonads WT: BW11M1 wild type; xtlA, xtlB, xtlC, and xtlR: mutants CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively; xltR+: mutant CMPG2201 with pCMPG6204 containing xtlR of P. putida BW11M1. The phenotypes shown for the selected xtlA, xtlB and xtlC mutants are representative for the other xtl NRPS mutants (Table S1). The quantitative data for biofilm formation are shown in Fig. 5. doi:10.1371/journal.pone.0062946.g004 May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 6 Antimicrobial Pseudomonas putida Lipopeptide Antimicrobial Pseudomonas putida Lipopeptide The analysis of variance (ANOVA) test was used to evaluate significant differences (p,0.001; indicated with different letters above the bars) between the wild type (set to 100%; blue), mutants (red), and complemented regulatory mutant (green). doi:10.1371/journal.pone.0062946.g005 downstream oprM-like gene xtlF, encoding the presumed outer membrane component of the tripartite transport system, possibly diminishing xantholysin export. A complementation construct carrying both xtlR and xtlF could not be obtained to test this hypothesis. Xantholysin A: a Novel Cyclic Lipotetradecadepsipeptide LC-MS analysis of semi-purified extracts revealed the presence of four lipopeptide compounds with molecular masses 1775.4 Da (main compound, xantholysin A), 1761.2 Da (minor variant 1, xantholysin B), 1802.0 Da (minor variant 2, xantholysin C) and 1775.2 Da (minor unidentified xantholysin variant 3, xantholysin D) (Fig. S6, Fig. S7). Only xantholysin A could be successfully isolated in sufficient quantity to allow adequate NMR analysis (Fig. S8, Table S6). The modest quantities obtained for the minor xantholysin variants did not allow to record NMR data of sufficient quality for a de novo analysis and full spectral assignment (Fig. S12, Fig. S13). Nevertheless, direct comparison of the 1H-1H TOCSY and 1H-13C HSQC spectra for xantholysin B and C with the corresponding spectra measured on xantholysin A allowed to identify the nature of the variation. For both compounds, most of the 1H and 13C chemical shifts were nearly identical to xantholysin A, demonstrating their overall high similarity. In the case of xantholysin B, the single observed difference is a substitution of the Ile14 spin system for a Val spin system, explaining the 14 Da mass difference (Fig. S14). The spectra indicated the lipid tail moiety of xantholysin C to consist of 3-hydroxydodec-5-enoate (Fig. S13), accounting for the 26 Da mass increase. High-resolution mass spectra of both compounds confirmed the chemical formulae to be C83H144N18O23 (exact mass: 1761.0574 Da) and C86H148N18O23 (exact mass: 1801.0900 Da) for xantholysin B and C, respectively (Fig. S15, Fig. S16). Considerations of the 13C chemical shifts within the lipid tail moiety of xantholysin C suggest the double bond to be in the cis configuration (Fig. S17). This is reminiscent of the promiscuous lipoinitiatory activity reported for Pseudomonas corrugata producing similar amounts of corpeptin A (HDA) and corpeptin B (3-hydroxydodec-5-enoic acid) [62]. In most lipopep- tide-producing Pseudomonas, promiscuity is observed with respect to chain length of the incorporated saturated 3-hydroxy fatty acids. Antimicrobial Pseudomonas putida Lipopeptide Figure 5. Biofilm formation by P. putida BW11M1 and xantholysin-deficient mutants. Abbreviations as in Fig. 4. Error bars indicate standard deviations. The analysis of variance (ANOVA) test was used to evaluate significant differences (p,0.001; indicated with different letters above the bars) between the wild type (set to 100%; blue), mutants (red), and complemented regulatory mutant (green). doi:10.1371/journal.pone.0062946.g005 spectrum confirmed the chemical formula as C84H146N18O23 (exact mass: 1775.0764 Da; Fig. S11), thus indirectly providing additional support for the number of Gln and Glu residues and the length (C10) of the fatty acid chain. At this point, the only feature of the proposed xantholysin A structure which still remained to be confirmed was the depsi bond between Ile14 and Ser7. The predicted molecular mass of the above sequence when considering it as a linear lipopeptide deviates 18 Da with the experimental value, in agreement with the expected presence of the lactone bond. Based on previous experience in assigning cyclic lipodepsipeptides [60], the only direct demonstration of the existence of this bond would be by detecting a 3JCH correlation between the C-terminal Ile14 carbonyl and the Ser7 CH2 b resonances in the HMBC spectrum. While spectral overlap prevented convincing observation of such correlation, indirect but clear evidence of the presence of the depsi bond is found in the chemical shift of the Ser7 CH2 b protons, which is significantly higher (4.38 ppm and 4.58 ppm) compared to their random coil values (3.79 ppm and 3.95 ppm) [61]. A similar downfield chemical shift for protons neighboring the depsi bond is observable for other cyclic lipodepsipeptides as well [60] and is explained by an anisotropic deshielding effect created by the carbonyl double bond. This phenomenon can thus be seen as a characteristic feature for this class of compounds, allowing quick recognition of the presence and location of the depsi bond. Importantly, the only other chemically feasible location of the depsi bond – the alcohol group of the HDA moiety – can in this way be excluded, since the HDA Hb resonance does not experience such a strong downfield shift in comparison to other cyclic lipodepsipeptides that were assigned by NMR [17,60]. Figure 5. Biofilm formation by P. putida BW11M1 and xantholysin-deficient mutants. Abbreviations as in Fig. 4. Error bars indicate standard deviations. The Broad Antifungal Activity of P. putida BW11M1 is mainly Dependent on Xantholysin Production To further assess the antimicrobial potential of xantholysin, the capacity of P. putida BW11M1 and its xantholysin-deficient mutants to inhibit growth of several fungi was compared. Radial mycelial outgrowth was delayed or arrested for most of the fungi confronted with a colony of wild-type BW11M1 and this was connected to xantholysin production (Fig. S5). In addition to The lack of full complementation of the regulatory mutant to wild-type level of biofilm formation, also observed to a certain extent for antagonistic and hemolytic phenotypes, may be due to some polar effect of the xtlR insertion on expression of the May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org May 2013 \| Volume 8 \| Issue 5 \| e62946 7 Antimicrobial Pseudomonas putida Lipopeptide Antimicrobial Pseudomonas putida Lipopeptide Relaxed amino acid substrate specificity, as observed here for the terminal module in XtlC giving rise to minor amounts of xantholysin B, is common among Pseudomonas NRPS systems [5,8,9,43]. ( g , ) The 2D 1H-1H TOCSY spectrum of xantholysin A in DMF-d7 (Fig. S9) allowed the identification of residues corresponding to five Leu, one Ile, two Val, five Glx and one Ser by means of their characteristic amino acid correlation patterns. The presence of a 3-hydroxydecanoic acid (HDA) moiety was determined from combined analysis of the 1H-1H TOCSY and 1H-13C HSQC spectra and comparison with the chemical shifts reported for pseudodesmin A in dimethylformamide solution [17,60]. The sequence (HDA-Leu1-Glx2-Glx3-Val4-Leu5-Glx6-Ser7-Val8- Leu9-Glx10-Leu11-Leu12-Glx13-Ile14) was established by ob- serving sequential Ha-HN cross-peaks in the 2D 1H-1H NOESY spectrum (Fig. S10). The TOCSY and NOESY spectra also revealed the presence of four individual pairs of mutually correlated amide resonances originating from primary amide functional groups. Correlations in the NOESY spectrum between these resonances and the CH2 c and CH2 b resonances of the Glx residues allowed specific assignment of each amide resonance pair to a Glx residue, effectively establishing the identity of residues 3, 6, 10 and 13 to be Gln. The absence of any such correlations to the CH2 c and CH2 b resonances of residue 2 and any further primary amide group resonance pair in the spectrum established the identity of residue 2 as Glu. The 1H-13C HMBC spectrum allowed the assignment of several 13C carbonyl resonances from the 2JCH coupling with the HN resonances (Table S6). The resulting peptide sequence (Leu-Glu-Gln-Val-Leu-Gln-Ser-Val- Leu-Gln-Leu-Leu-Gln-Ile; Fig. 2) largely confirmed the phyloge- ny-based prediction using an organism-specific subset of experi- mentally confirmed A-domain sequences. A high-resolution mass Xantholysin-like lipopeptides with antiviral activity were isolat- ed from Pseudomonas sp. RtIB026. The main compound MA026 (PubChem CID 10285742) differs by two consecutive amino acids (Leu10-Gln11 in MA026 versus Gln10-Leu11 in xantholysin A). Congener R1MA026 (with Val9-Leu14) contains an additional May 2013 \| Volume 8 \| Issue 5 \| e62946 May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 8 Antimicrobial Pseudomonas putida Lipopeptide striking for the initiatory enzymes XtlA and EtlA, both composed of two modules, and the subsequently acting enzymes, XtlB and EtlB. Overall, the latter enzymes share eight probably equifunc- tional modules but a single differently positioned serine-incorpo- rating unit generates a different amino acid sequence stretch. Materials and Methods Bacterial Strains, Plasmids and Culture Conditions Bacterial strains and plasmids used are described in Table 1 and Table S4. TSB growth medium (trypticase soy broth, 30 g/L; BD Biosciences) was used for Pseudomonas and Xanthomonas strains at 30uC and for Klebsiella pneumoniae ATCC 13883 and Staphylococcus aureus ATCC 6358 at 37uC. Aeromonas hydrophila ATCC 7966, Agrobacterium tumefaciens A208, Azospirillum brasilense Sp7, Burkholderia vietnamiensis LMG 10927, Mycobacterium smegmatis DSM 43756, Rhodococcus erythropolis N11, Serratia entomophila DSM 12358, Sphingomonas wittichii RW1, Variovorax paradoxus LMG 1797 and Antimicrobial Pseudomonas putida Lipopeptide Furthermore, this apparent ‘‘serine switch’’ also shifts the site of macrocyclization with three residues, such that xantholysin carries an octacyclic moiety instead of the pentacyclic structure in entolysin. Although the three NRPS enzymes from strains BW11M1 and L48 share relatively high pairwise sequence homology (70.5% amino acid identity for ,15,440 concatenated residues), the architectural divergence engenders synthesis of quite different molecules that can be assigned to separate groups within the Pseudomonas lipopeptide classification, based on amino acid sequence (length and similarity) and presence/type of macrocycle [4–6]. switch of two residues compared to xantholysin B (with Leu9- Val14). Congener R2MA026 differs from MA026 by a 3- hydroxydodec-5-enoyl instead of a 3-hydroxydecanoyl fatty acid moiety (same as the difference between xantholysins A and C). Given these striking similarities, it would be of interest to identify the biosynthetic genes of strain RtIB026 (patent strain FERM BP- 7436) to compare the modular structure of its NRPSs with those of the xantholysin system. The MA026 structure suggests a peculiar A-domain switch between the last module of the second enzyme and the first module of the third enzyme in RtIB026, compared to XtlB and XtlC, respectively. Given the phylogenetic separation of the Val- and Leu-selective A-domains of module 9 and 14 in xantholysin (Fig. 3), the biosynthesis of an as yet unidentified Val9- Leu14 congener by the xantholysin synthetases is unlikely. Since for none of these RtIB026 structures a detailed NMR analysis is available that would allow comparison to the xantholysins, it is at this point still unclear whether indeed such striking biosynthetic variation is occurring or whether the RtIB026 structures require revision. Unexpected Similarities among Pseudomonas NRPS Systems: the Advantage of being Modular Phylogenetic comparison of the respective NRPS domains, the linked LuxR-family regulator and the associated tripartite export system consistently points to evolutionary relatedness of the components of the xantholysin and entolysin systems with those of P. putida strains generating lipopeptides that – at first glance – show little structural similarity: putisolvin (twelve amino acids, tetracyclic; [16]) and WLIP (nine amino acids, heptacyclic; [17]). These four P. putida lipopeptides share a very similar A-domain pair in the initiatory synthetases (EtlA, PsoA, WlpA, XtlA; Fig. 6). The equivalent modules in the P. fluorescens starter enzymes for incorporation of the same dipeptide (Leu-Glu) in viscosin-group lipopeptides cluster separately from the P. putida ones [18]. From Fig. 6 it is also apparent that xantholysin synthetase B shares a core of five phylogenetically related contiguous modules with putisolvin synthetase B (A-domains for Leu/Ile-Gln-Ser-Val-Leu/ Ile). Comparison of the A-module architecture of entolysin, WLIP and putisolvin synthetases C points to a tripartite terminating block composed of A-domains with common evolutionary origin, but arranged in a different order in PsoC compared to EtlC and WlpC. Distinct Antibiotic Properties of Xantholysin Distinct Antibiotic Properties of Xantholysin The identification of xantholysin adds to the growing list of cyclic lipopeptides produced by pseudomonads. Like several other Pseudomonas lipopeptides [3], xantholysin appears to promote surface colonization of its producer, P. putida BW11M1, a strain isolated from the rhizosphere of banana plants. However, unlike most pseudomonad lipopeptides, antimicrobial activity is not confined to fungi and Gram-positive bacteria, but also extends to some Gram-negative strains, including several xanthomonads. Members of this c-proteobacterial genus that are colonizing similar plant niches as pseudomonads [29–32], are also susceptible to WLIP, a member of the viscosin group [17], but Xanthomonas strains exhibit differences in sensitivity to both lipopeptides. During this study it became evident that, in addition to xantholysin, strain BW11M1 produces one or more as yet unidentified antimicrobial factor(s), also affecting a number of xanthomonads. While xantholysin does not exert a growth- inhibitory effect on fellow pseudomonads, P. putida BW11M1 also produces a plant lectin-like bacteriocin to target other pseudomo- nads, including members of the P. syringae group [35,63], indicating that both ribosomally and non-ribosomally synthesized antimicrobials are part of its armory deployed against competitors. Antagonism among plant-associated c-proteobacterial relatives has been described for some other types of pseudomonad secondary metabolites: the modified amino acid 3-methylarginine causing inhibition between P. syringae pathovars [64]; the salicylate- containing P. putida antibiotic promysalin, targeting other pseudo- monads [65]; oxyvinylglycines active against Erwinia amylovora [66,67]. In view of the structural similarity of xantholysins to certain pseudomonad lipopeptides with antiviral activity (viz. MA026 and its congeners), future experiments should also address the effects of xantholysins on enveloped viruses. Antiviral activity has been reported for some other lipopeptides, such as viscosin and surfactin [3]. Collectively, these observations suggest that these pseudomo- nads probably have recruited and reshuffled gene modules to engineer novel NRPS assembly lines for synthesis of new structural lipopeptide variants. In combination with rapidly accumulating genomic sequence data, high-throughput exploration of this part of the ‘‘parvome’’ [68] becomes feasible with recently developed strategies of mass spectral metabolic profiling of live microbial colonies [44,69]. In addition to identification of new molecules and associated biological activities, this will likely provide insight in the extent of modular diversity among these biosynthetic enzymes, and how this has been generated and further evolved in prominent lipopeptide producers such as Pseudomonas and Bacillus. General Conclusions Distinct Antibiotic Properties of Xantholysin Xantholysin and Entolysin Synthetases: Similar Enzymes but Very Different Products Xantholysin and Entolysin Synthetases: Similar Enzymes but Very Different Products The genetic backbone of the xantholysin NRPS system bears considerable similarity to the one for entolysin biosynthesis by the P. putida relative P. entomophila L48 [10], both consisting of a fourteen-unit assembly line with three enzymes operating in co- linear mode (Fig. 6). Conservation of modular architecture is most May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 9 Antimicrobial Pseudomonas putida Lipopeptide Figure 6. Modular architecture of the xantholysin synthetases and other Pseudomonas enzymes with similar A domains. The respective starter NRPS genes are located distantly from the gene pairs encoding the middle and terminating NRPSs, except for the putisolvin operon psoABC [16]. Synthesis of the peptide moiety by the consecutive action of the modules (with only A domains shown) proceeds in a co-linear fashion, with incorporation of the respective amino acid at the positions corresponding to the numbered boxes. If a minor lipopeptide variant with a different amino acid at a certain position has been identified, this residue is shown in parentheses with smaller font. Xle indicates that the residue’s identity (either Leu or Ile) was not resolved in putisolvin II. The connected residues form a depsi bond. Similarity between the different systems is visualized according to Rokni-Zadeh et al. [18]: two domains with a patristic distance ,0.45 (summed branch lengths in a maximum-likelihood tree constructed from aligned A-domain sequences; see Fig. 3) are represented in the same color. doi:10.1371/journal.pone.0062946.g006 Figure 6. Modular architecture of the xantholysin synthetases and other Pseudomonas enzymes with similar A domains. The respective starter NRPS genes are located distantly from the gene pairs encoding the middle and terminating NRPSs, except for the putisolvin operon psoABC [16]. Synthesis of the peptide moiety by the consecutive action of the modules (with only A domains shown) proceeds in a co-linear fashion, with incorporation of the respective amino acid at the positions corresponding to the numbered boxes. If a minor lipopeptide variant with a different amino acid at a certain position has been identified, this residue is shown in parentheses with smaller font. Xle indicates that the residue’s identity (either Leu or Ile) was not resolved in putisolvin II. The connected residues form a depsi bond. Similarity between the different systems is visualized according to Rokni-Zadeh et al. Xantholysin and Entolysin Synthetases: Similar Enzymes but Very Different Products [18]: two domains with a patristic distance ,0.45 (summed branch lengths in a maximum-likelihood tree constructed from aligned A-domain sequences; see Fig. 3) are represented in the same color. doi:10.1371/journal.pone.0062946.g006 Yersinia enterocolitica LMG 7899 were grown at 30uC, and Bordetella avium 197N, E. coli and Salmonella enteritidis ATCC 13076 at 37uC, in LB (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L). Nutrient broth (8 g/L) was prepared for growing Bacillus megaterium ATCC 13632 and Erwinia amylovora CFBP 1430 (at 30uC), and for Citrobacter freundii ATCC 8090, Enterobacter aerogenes ATCC 13048, Proteus vulgaris LMM 2011 and Shigella flexneri LMG 10472 (at 37uC). YEP (yeast extract 10 g/L, bacterial peptone 10 g/L) was used for Burkholderia cepacia LMG 1222, TY (tryptone 5 g/L, yeast extract 3 g/L, CaCl2 7 mM) for Bacillus subtilis LMG 7135 (all grown at 30uC), and MRS (Difco lactobacilli MRS broth 55 g/L) for Lactobacillus plantarum LMG-P21295 (cultured at 30uC) and Lactobacillus rhamnosus GG (LMG 6400, at 37uC). Streptomyces coelicolor A3(2) and Streptomyces lividans TK24 were cultivated in a medium composed of 10 g casitone, 5 g yeast extract and 5 g glucose per liter at 30uC. For solid media 15 g per liter agar was added. Antibiotics were added at the following concentration when required: kanamycin (50 mg/ml), ampicilin (100 mg/ml), tetracycline (20 mg/ml). culture (,108–109 CFU/ml) and poured onto the plates as an overlay. Following day, plates (three replicates) were evaluated for growth inhibition halos in a turbid lawn of indicator cells. Protease sensitivity of the antimicrobial activity was verified by spotting 10 ml of proteinase K (20 mg/ml) near a chloroform-killed producer colony prior to overlay. Yersinia enterocolitica LMG 7899 were grown at 30uC, and Bordetella avium 197N, E. coli and Salmonella enteritidis ATCC 13076 at 37uC, in LB (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L). Nutrient broth (8 g/L) was prepared for growing Bacillus megaterium ATCC 13632 and Erwinia amylovora CFBP 1430 (at 30uC), and for Citrobacter freundii ATCC 8090, Enterobacter aerogenes ATCC 13048, Proteus vulgaris LMM 2011 and Shigella flexneri LMG 10472 (at 37uC). Xantholysin and Entolysin Synthetases: Similar Enzymes but Very Different Products YEP (yeast extract 10 g/L, bacterial peptone 10 g/L) was used for Burkholderia cepacia LMG 1222, TY (tryptone 5 g/L, yeast extract 3 g/L, CaCl2 7 mM) for Bacillus subtilis LMG 7135 (all grown at 30uC), and MRS (Difco lactobacilli MRS broth 55 g/L) for Lactobacillus plantarum LMG-P21295 (cultured at 30uC) and Lactobacillus rhamnosus GG (LMG 6400, at 37uC). Streptomyces coelicolor A3(2) and Streptomyces lividans TK24 were cultivated in a medium composed of 10 g casitone, 5 g yeast extract and 5 g glucose per liter at 30uC. For solid media 15 g per liter agar was added. Antibiotics were added at the following concentration when required: kanamycin (50 mg/ml), ampicilin (100 mg/ml), tetracycline (20 mg/ml). Growth Inhibition of Fungi An agar plug with fungal mycelium, grown for 1–2 weeks on half-strength PDA (potato dextrose agar) medium was placed at the center of an agar plate with appropriate medium at 20uC. PTA medium (potato dextrose broth 12 g, TSB 3 g, agar 15 g per liter) plates were used for most fungi. Alternaria porri was grown on 6CA- TSB medium (Bambix 6 cereals 10 g, TSB 15 g, agar 15 g per liter). 5 ml of overnight cell culture of P. putida BW11M1 and selected xantholysin-deficient mutants were spotted at a distance of ,3 cm from the inoculum plug and incubated at 20uC for several days up to two weeks, depending on the growth rate of the fungi, before assessing the occurrence of mycelial growth inhibition zones. Indicator fungi are listed in Table S5. Antibacterial Activity Assays by Agar Diffusion The effect of xantholysin production on Agaricus bisporus was carried out as described by Bessette [52] with minor modifications. Briefly, 20 ml of an overnight Pseudomonas culture (,108– 109 CFU/ml) was spotted onto horizontally sliced pilei of mushroom basidiocarps (Agaricus bisporus). After absorption of the liquid by the sterile surface, mushrooms were incubated overnight at 30uC, sealed in a closed plastic box and saturated with water to prevent drying out. Following day, mushrooms were scored for the P. putida BW11M1 was grown in 5 ml TSB for 8 h with shaking (200 rpm). Two ml of this culture (OD600 ,1.8) was spotted onto a TSB agar and incubated overnight at 30uC. After exposure to chloroform vapor for 30 min to kill producer bacteria, the plates were opened in a laminar flow chamber to evaporate residual chloroform during another 30 min. Three ml of molten, semisolid agar (0.5%) was inoculated with 100 ml of a bacterial indicator cell May 2013 \| Volume 8 \| Issue 5 \| e62946 May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 10 Antimicrobial Pseudomonas putida Lipopeptide Table 1. Bacterial strains and plasmids. Strain or plasmid Description and characteristicsa Source or Reference Strains E. coli TOP10F’ F9{lacIq, Tn10(TetR)} mcrA D(mrr-hsdRMS-mcrBC) W80lacZDM15 DlacX74 recA1 araD139 D(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG Invitrogen E. coli EPI300-T1R Host strain for pCC2FOS derivatives Epicentre Biotechnologies E. coli DH5a supE44 DlacU169 (W80 lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 [70] E. coli HB101 (pRK2013) Helper strain in triparental conjugation, Kmr [71] P. putida BW11M1 Banana rhizosphere (Sri Lanka) [33] P. putida CMPG2183 BW11M1 xtlA mutant This study P. putida CMPG2187 BW11M1 xtlB mutant This study P. putida CMPG2198 BW11M1 xtlC mutant This study P. putida CMPG2201 BW11M1 xtlR mutant This study X. alfalfae subsp. alfalfae LMG 497 Pathovar reference strain; Medicago sativa (Sudan) BCCMb X. axonopodis pv. manihotis LMG 784 Pathovar reference strain; Manihot esculenta (Brazil) BCCM X. campestris pv. campestris LMG 582 Brassica sp. (Belgium) BCCM X. citri pv. malvacearum LMG 761 Pathovar reference strain; Gossypium sp. (Sudan) BCCM X. hortorum pv. hederae LMG 7411 Hedera helix (USA) BCCM X. sacchari LMG 471 Type strain; Saccharum officinarum (Guadeloupe) BCCM X. sp. pv. zinniae LMG 8692 Pathovar reference strain; Zinnia elegans (Australia) BCCM X. translucens LMG 12921 Anthurium andreanum (USA) BCCM X. translucens pv. Antibacterial Activity Assays by Agar Diffusion cerealis LMG 679 Pathovar reference strain; Bromus inermis (USA) BCCM X. translucens pv. graminis LMG 726 Pathovar reference strain; Dactylis glomerata (Switzerland) BCCM X. translucens pv. hordei LMG 737 Pathovar reference strain; Hordeum vulgare (India) BCCM X. vasicola pv. holcicola LMG 736 Pathovar reference strain; Sorghum bicolor (New Zealand) BCCM X. vasicola pv. musacearum LMG 785 Pathovar reference strain; Ensete ventricosum (Ethiopia) BCCM X. vasicola pv. musacearum LMG 7431 Musa sp. (Ethiopia) BCCM X. oryzae pv. oryzae PXO99 Race 6; Oryza sativa (The Philippines) IRRIc X. oryzae pv. oryzae PXO112 Race 5; Oryza sativa (The Philippines) IRRI X. oryzae pv. oryzae PXO340 Race 3; Oryza sativa (The Philippines) IRRI Plasmids pCC2FOS Copy control fosmid Epicentre Biotechnologies pJB3Tc20 Broad-host-range cloning vector, Apr, Tcr [72] pCMPG6126 Fosmid clone containing xtlA This study pCMPG6127 Fosmid clone 12H4 containing xtlB and partial xtlC This study pCMPG6128 Fosmid clone 19F2 containing xtlC and partial xtlB This study pCMPG6204 pJB3Tc20 containing xtlR, Tcr This study Other bacteria used as indicator strains are listed in Table S4. aAntibiotic resistance phenotypes: Apr, ampicillin resistance, Kmr, kanamycin resistance; Tcr, tetracycline resistance. bBCCM, Belgian Coordinated Collections of Microorganisms. cIRRI International Rice Research Institute Description and characteristicsa Source or Reference Other bacteria used as indicator strains are listed in Table S4. aAntibiotic resistance phenotypes: Apr, ampicillin resistance, Kmr, kanamycin resistance; Tcr, tetracycline resistance. bBCCM, Belgian Coordinated Collections of Microorganisms. cIRRI, International Rice Research Institute. doi:10.1371/journal.pone.0062946.t001 identified by the plasmid rescue method [36] and subsequent sequencing of the flanking regions using pTnMod-OKm’-specific primers PGPRB-0293 (59-TCTGGCTGGATGATGGGGCG-39) and PGPRB-0294 (59-CGGTTCCTGGCCTTTTGCTGGC - 39). presence of browning. Cultures of wild type and mutants were tested in parallel (three independent biological repeats). Isolation and Characterization of Mutants with Abolished Anti-Xanthomonas Activity A BW11M1 plasposon pTnMod-OKm’ mutant library [35] was screened for mutants with abolished activity against indicator strain X. alfalfae subsp. alfalfae LMG 497 by the agar diffusion method. Each mutant from a 96-well microtiter plate was stamped onto TSB agar square petri dishes (12612 cm) with a 96-prones replicator. Mutants lacking anti-LMG 497 activity were selected and confirmed twice. The plasposon-interrupted genes were Fosmid Library Construction and Screening Fosmid Library Construction and Screening For construction of a P. putida BW11M1 genomic library in fosmid pCC2FOS, the CopyControl HTP Fosmid Library Production Kit protocol (Epicentre Biotechnologies) was applied. Based on the flanking sequences identified in the plasposon mutants, pairs of primers were designed to screen for fosmid clones May 2013 \| Volume 8 \| Issue 5 \| e62946 11 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Antimicrobial Pseudomonas putida Lipopeptide water (pH 3). Methanol was used to dissolve the pellet and the solution was kept at 4uC till further purification. After evaporation of solvent, the slurry was extracted with cold methyl tert-butyl ether to precipitate the peptide fraction. This procedure was repeated three times to obtain a crude peptide-rich mixture which was further purified by preparative RP-HPLC. Samples were dissolved in methanol (50 mg/ml) and eight injections of 1 ml were performed on a Phenomenex column (Luna C18(2) 250621.2 mm, 5 mm particle size) operating at a flow rate of 17.5 ml/min. The ratio of the mobile phase solvents, 5 mM ammonium acetate in water and acetonitrile, was changed in a linear fashion from 50:50 to 10:90 over a time span of 15 minutes. The major compound (xantholysin A) was collected based on UV detection at l = 214 nm. Two additional minor lipopeptides, shown to be xantholysin variants B and C, were isolated by analytical HPLC using an Agilent 1100 series instrument equipped with a Phenomenex column (Luna C18(2), 25064.6 mm, 5 mm particle size) and operating at a flow rate of 0.8 ml/min. The ratio of mobile phase solvents, 5 mM ammonium acetate in water and acetonitrile, was changed from 35:65 to 25:75 over a time span of 15 minutes. To identify bioactive fractions, samples from collected fractions were spotted on a TSB agar plate and overlayed with indicator cells after evaporation of the solvent (with non-polar phase solvent as negative control). containing the corresponding genomic DNA regions by colony PCR. Selected clones were compared by end-sequencing using pCC2FOS-specific primers PGPRB-1319 (59-GTACAACGA- CACCTAGAC-39) and PGPRB-1320 (59-CAGGAAACAGCC- TAGGAA-39). Finally, three fosmids with DNA fragments covering the genomic regions of interest were sequenced at Macrogen (Korea). The insert size of the fosmids was estimated by separation of NotI-generated fragments using pulsed-field gel electrophoresis. Bioinformatic Analyses Domains were identified using NRPS analysis tools [73,74]. The NRPSpredictor2 tool was used to predict amino acid specificity of A domains [38]. Amino acid sequences were aligned with MUSCLE for maximum-likelihood tree construction using (PhyML with JTT matrix; [75]) as implemented in Geneious Pro (version 5.6.3). Fosmid Library Construction and Screening A high copy number of the fosmids was induced by treating cells with the CopyControl Induction solution, prior to extraction of the fosmids with the FosmidMAX DNA purification kit (Epicentre Biotechnologies). The contigs with the regions containing the functionally characterized genes were submitted to GenBank (accession numbers KC297505 and KC297506). Complementation of xtlR Regulatory Mutant Complementation of mutant CMPG2201 was performed as previously described [17]. Briefly, the xtlR gene was amplified using Platinum Pfx DNA polymerase (Invitrogen) with primers PGPRB-8013 (59-TTGCTCTAGATCAGGTGCCGGCCATC- CAGC-39) and PGPRB-8014 (59-CTCGGAATTCGGATCAAG- GAAGACGACGAC-39) and cloned into the pJB3Tc20 vector using the XbaI and EcoRI sites. The resulting construct (pCMPG6204) was transferred to E. coli DH5a. Conjugative transfer of pCMPG6204 to P. putida BW11M1 mutant CMPG2201 was achieved by triparental conjugation using E. coli HB101 bearing pRK2013 as helper strain. The phenotypes of a selected transconjugant were compared to the regulatory mutant. Swarming Motility, Biofilm Formation and Hemolytic Activity High-resolution mass spectra were recorded on an Agilent 6220A time-of-flight mass spectrometer, equipped with an Agilent ESI/APCI multimode source. The ionization mode was set to APCI (atmospheric pressure chemical ionization), while the mass spectra were acquired in 4 GHz high-resolution mode with a mass range set to 3200 Da. All NMR measurements on xantholysin A were performed on a sample of 6.5 mg dissolved in 600 ml DMF- d7 (Eurisotop). High quality HP-7 (New Era Ent. Inc) NMR tubes were used. NMR experiments were performed on a Bruker Avance III spectrometer operating at 500.13 MHz and 125.76 MHz for 1H and 13C, respectively, and equipped with a 5 mm 1H BBI-Z probe. Due to the low absolute quantities of compound after purification, the xantholysin variants were dissolved in 10 ml DMF-d7 for measurements using 1 mm capillaries. In this case, NMR experiments were performed on a Bruker Avance II spectrometer operating at 700.13 MHz and 176.04 MHz for 1H and 13C, respectively, and equipped with a 1 mm 1H, 13C, 15N TXI-Z probe. For both analysis of xantholysin A and its variants, sample temperature was set to 55uC throughout, as significant line broadening effects occurred at lower temperatures. 2D spectra measured for structure elucidation include a 1H-1H TOCSY with a 90 ms MLEV-17 spinlock, a sensitivity-improved, multiplicity edited, 1H-13C gHSQC applying adiabatic 180u pulses, a 1H -1H NOESY with a 300 ms mixing time and a 1H-13C gHMBC experiment optimized for a carbon- Swarming ability was studied by spotting 2 ml of 8 h-grown cells on TSB medium solidified with 0.8% agar and evaluating surface spreading after incubation at 30uC for 16 to 24 h (three repeats for each sample). Biofilm formation was assayed according to Janssens et al. [76] by using flat-bottom 96-well microtiter plates and polystryrene pegs (Nunc, Sanbio b.v.). Cells grown for 8 hours were adjusted to OD600 ,1.0 and then diluted 100-fold as initial culture (200 ml per well, 8 wells per strain). After 2 days of incubation at 30uC, biofilm formation was visualized by staining pegs with 0.1% crystal violet. Stained biofilms were subsequently extracted with 30% acetic acid and quantified by measuring absorbance of the solution at 570 nm. Hemolytic activity was assayed by spotting 2 ml of overnight-grown cells on TSB agar with 5% horse blood (Oxoid) plates and hemolysis halos were examined after incubating plates overnight at 30uC. Xantholysin Structure Elucidation Initial mass spectra were acquired on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3 ml/min and spectra were obtained in positive ionization mode with a resolution of 15000 FWHM and accuracy of 0.5 ppm RSD using leucine enkephalin as lock mass. Additional LC-MS data were collected on the extracted mixture prior to purification with an Agilent 1100 Series HPLC with an ESI detector type VL, equipped with a Phenomenex column (Luna C18(2), 25064.60 mm, 5 mm particle size). The flow rate was 1 ml/min. The mobile phase solvents, 5 mM ammonium acetate in water and acetonitrile, were linearly changed from a 25:75 ratio to a 0:100 ratio over a time span of 15 minutes. Lipopeptide Extraction and Purification Overnight TSB-grown BW11M1 cell culture was used to inoculate fresh TSB medium at a volume ratio of 1:100 and incubated at 30uC for at least 2 days with shaking (200 rpm). After removing cells through centrifugation (6370 g, 30 min, 4uC), the supernatant was acidified by adding 9% HCl to lower the pH to pH 2–3. The precipitate formed was isolated by centrifugation (6370 g, 30 min, 4uC) and washed twice with acidified distilled May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 12 Antimicrobial Pseudomonas putida Lipopeptide deficient mutants (xtlA, xtlB, xtlC, xtlR; CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively). The application pattern of bacterial cell suspensions spotted around a centrally positioned plug with fungal mycelium is shown schematically in (A). (B) Alternaria longipes CBS 620.83, (C) Ascochyta pisi MUCL30164, (D) Botrytis cinerea MUCL30158, (E) Colletotrichum gloeosporoides SR24, (F) Fusarium culmorum MUCL30162, (G) Fusarium graminearum MUCL30161, (H) Fusarium oxysporum MUCL909, (I) Fusarium oxysporum MUCL30160, (J) Fusarium oxysporum f. sp. radices-lycopersi CBS 873.95, (K) Fusarium oxysporum f. sp. vasinfectum CBS 410.90, (L) Gloeosporium musarum MUCL500, (M) Gloesporium solani CBS 194.32, (N) Nectria haematococca MUCL20259, (O) Pyrenophora tritici-repentis MUCL30217, (P) Rhizoctonia solani CBS 211.84. (PDF) deficient mutants (xtlA, xtlB, xtlC, xtlR; CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively). The application pattern of bacterial cell suspensions spotted around a centrally positioned plug with fungal mycelium is shown schematically in (A). (B) Alternaria longipes CBS 620.83, (C) Ascochyta pisi MUCL30164, (D) Botrytis cinerea MUCL30158, (E) Colletotrichum gloeosporoides SR24, (F) Fusarium culmorum MUCL30162, (G) Fusarium graminearum MUCL30161, (H) Fusarium oxysporum MUCL909, (I) Fusarium oxysporum MUCL30160, (J) Fusarium oxysporum f. sp. radices-lycopersi CBS 873.95, (K) Fusarium oxysporum f. sp. vasinfectum CBS 410.90, (L) Gloeosporium musarum MUCL500, (M) Gloesporium solani CBS 194.32, (N) Nectria haematococca MUCL20259, (O) Pyrenophora tritici-repentis MUCL30217, (P) Rhizoctonia solani CBS 211.84. (PDF) proton coupling of 8 Hz. Standard pulse sequences as present in the Bruker library were used throughout. Typically, 2048 data points were sampled in the direct dimension for 256 data points in the indirect one, with the spectral width respectively set to 12 ppm along the 1H dimension and 110 ppm (gHSQC) or 220 ppm (gHMBC) along the 13C dimension. For 2D processing, the spectra were zero filled to obtain a 204862048 real data matrix. Supporting Information Figure S1 Cladogram representation of a maximum- likelihood tree inferred from amino acid sequence alignment of condensation domains extracted from characterized Pseudomonas NRPSs. The lipopeptide-spe- cific codes are specified in Fig. 3, with the xantholysin-related ones in bold. Clusters are differentiated according to the type of C- domain by color: conventional domains (green), dual epimeriza- tion/condensation domains (blue), and N-acylating starter do- mains (red). The tree was rooted with the divergent C1 domain of SyrE (in black). Figure S6 Chromatographic separation of xantholysin congeners. LC-MS chromatogram of the extracted mixture prior to purification using UV-detection at 214 nm. (PDF) Figure S7 Mass spectrometry of xantholysin congeners. Mass spectra of xantholysin A and its variants obtained during LC-MS analysis of the extracted mixture prior to purification. Preliminary experiments with a high-resolution instrument showed that the peaks represent ions with charge z = 2. (PDF) Figure S2 Maximum-likelihood tree of thioesterase domains of characterized LP-producing Pseudomonas NRPSs. The clusters with tandemly organized domains TE1 (red) and TE2 (green) are shown in different colors. Lipopeptide-specific codes as in Fig. 3, with the xantholysin-related ones in bold. The tandem-TE domains of lysobactin synthetase LybB from Lysobacter sp. ATCC 53042 and the mono-TE domain of syringomycin synthetase SyrE from P. syringae pv. syringae B301D are included for comparison. The internal residue involved in cyclization with the carboxyterminal amino acid is specified for each TE1/TE domain (NA = not applicable for the linear syringafactins; OH-Phe = b- hydroxy phenylalanine). The scale bar represents 0.5 substitutions per site. (PDF) Figure S8 1D NMR analysis of xantholysin A. 1D 1H spectrum of xantholysin A in DMF-d7 solution, 55uC, 500 MHz. (PDF) Figure S9 2D NMR analysis of xantholysin A. 2D 1H-1H TOCSY spectrum of xantholysin A in DMF-d7 solution, 55uC, 500 MHz, revealing the characteristic amino acid spin system patterns. (PDF) Figure S10 Nuclear Overhauser effect spectroscopy of xantholysin A. Established NOE contacts in xantholysin A, observed in a 2D 1H-1H NOESY spectrum with 300 ms mixing time. Figure S3 Comparison of cognate regulators and asso- ciated export systems in lipopeptide-synthesizing Pseu- domonas. Maximum-likelihood trees inferred from amino acid sequence alignments of (A) LuxR-family regulators encoded by genes linked with the respective initiatory NRPS genes and (B) the components of tripartite MacA/MacB/OprM-like export systems (concatenated sequences; same phylogeny was obtained for the three individual sequence sets (data not shown)). Lipopeptide- specific codes as in Fig. Lipopeptide Extraction and Purification Before Fourier transformation, all spectra were multiplied with a squared cosine bell function in both dimensions except for the gHMBC where a squared sine bell was applied. deficient mutants (xtlA, xtlB, xtlC, xtlR; CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively). The application pattern of bacterial cell suspensions spotted around a centrally positioned plug with fungal mycelium is shown schematically in (A). (B) Alternaria longipes CBS 620.83, (C) Ascochyta pisi MUCL30164, (D) Botrytis cinerea MUCL30158, (E) Colletotrichum gloeosporoides SR24, (F) Fusarium culmorum MUCL30162, (G) Fusarium graminearum MUCL30161, (H) Fusarium oxysporum MUCL909, (I) Fusarium oxysporum MUCL30160, (J) Fusarium oxysporum f. sp. radices-lycopersi CBS 873.95, (K) Fusarium oxysporum f. sp. vasinfectum CBS 410.90, (L) Gloeosporium musarum MUCL500, (M) Gloesporium solani CBS 194.32, (N) Nectria haematococca MUCL20259, (O) Pyrenophora tritici-repentis MUCL30217, (P) Rhizoctonia solani CBS 211.84. (PDF) Supporting Information 3, with the xantholysin-related ones in bold. The regulatory gene encoding SyrF (panel A) is located downstream of syrE. In panel B, the Syf and Arf systems are not included (no sequences available for the corresponding OprM-like protein). The scale bars represent 0.09 (panel A) and 0.04 (panel B) substitutions per site. The xantholysin cluster (blue) and viscosin cluster (green) are shown in different colors. (PDF) (PDF) Figure S11 High resolution mass spectrum of xantholy- sin A. (A) Full mass spectrum. (B) Zoom on the [M+H]+ molecular ion peaks. Expected exact mass of xantholysin A (C84H146N18O23)+H+: 1776.0881 Da; observed exact mass of xantholysin A+H+: 1776.0837 Da. (PDF) Figure S12 1D NMR analysis of xantholysin B. 1D 1H spectrum of xantholysin variant 1, in DMF-d7 solution, 55uC, 700 MHz. (PDF) Figure S13 1D NMR analysis of xantholysin C. 1D 1H spectrum of xantholysin variant 2, in DMF-d7 solution, 55uC, 700 MHz. The inset shows the signals arising from the alkene protons of the 3-hydroxydodec-5-enoic acid moiety, on top of an unidentified resonance from an impurity. (PDF) Figure S4 Growth inhibition of representative indicator bacteria by purified xantholysin. 10 ml-samples of purified xantholysin in methanol, containing 18 mg (A) or 9 mg (B) were spotted on an agar plate and overlaid with indicator cells after solvent evaporation. Methanol control spots did not cause growth inhibition. Figure S4 Growth inhibition of representative indicator bacteria by purified xantholysin. 10 ml-samples of purified xantholysin in methanol, containing 18 mg (A) or 9 mg (B) were spotted on an agar plate and overlaid with indicator cells after solvent evaporation. Methanol control spots did not cause growth inhibition. (PDF) Figure S14 2D NMR analysis of xantholysin B. 2D 1H-1H TOCSY spectrum of xantholysin variant 1, in DMF-d7 solution, 55uC, 700 MHz, revealing the characteristic amino acid spin system patterns. (PDF) Figure S5 Antifungal activity of xantholysin. Growth inhibition of fungi by P. putida BW11M1 (WT) and xantholysin- May 2013 \| Volume 8 \| Issue 5 \| e62946 PLOS ONE \| www.plosone.org 13 Antimicrobial Pseudomonas putida Lipopeptide plasposon mutants lacking antibacterial activity using X. alfalfae subsp. alfalfae LMG 497 as indicator strain. (PDF) Figure S15 High resolution mass spectrum of xantholy- sin B. (A) Full mass spectrum. (B) Zoom on the [M+H]+ and [M+Na]+ molecular ion peaks. Expected exact mass of xantholysin B (C83H144N18O23)+H+: 1762.0724 Da; observed exact mass of xantholysin B+H+: 1762.0647 Da. (PDF) Table S2 In silico analysis of adenylation domains in xantholysin synthetases. Predicted substrate specificity of the A domains in the xantholysin NRPSs. (PDF) Figure S16 High resolution mass spectrum of xantholy- sin C. (A) Full mass spectrum. (B) Zoom on the [M+H]+ and [M+Na]+ molecular ion peaks. Expected exact mass of xantholysin C (C86H148N18O23)+H+: 1802.1037 Da; observed exact mass of xantholysin C+H+: 1802.0973 Da. (PDF) (PDF) Table S3 Antagonistic activity spectrum of P. putida BW11M1 wild type (WT) and xantholysin-deficient mutants. The indicators shown are listed in Table 1 and Table S4. All other bacteria listed in Table S4 are not inhibited by strain BW11M1. Antagonism was semi-quantified by measuring the halo radii (in mm; average of three repeats; ND, no halo detected). Values in parentheses denote formation of a turbid halo. For some wild-type Xanthomonas strains a larger turbid halo was observed around a smaller clear halo. Additional italicized values in parentheses refer to the presence of a ‘secondary’ turbid growth inhibition halo. (PDF) Figure S17 NMR analysis of fatty acid moiety in xantholysin C. 1H and 13C chemical shift comparison between the lipid tail of xantholysin A and variant 2. (A) Experimental 1H (red) and 13C (blue) chemical shifts of the xantholysin 3- hydroxydecanoic acid moiety, compared to predicted 13C chemical shifts (green). (B) Experimental 1H (red) and 13C (blue) chemical shifts of the xantholysin variant 2 3-hydroxydodec-5- enoic acid moiety, compared to predicted 13C chemical shifts (green) assuming a cis-configuration. (C) Predicted 13C (green) chemical shifts of the xantholysin variant 2 3-hydroxydodec-5- enoic acid moiety assuming a trans-configuration. 13C chemical shift predictions were calculated using ChemNMR Pro 12.0 as implemented in the ChemDraw Ultra 12.0 software (PerkinElmer, Inc.). In the case of cis-3-hydroxydodec-5-enoic acid, the predicted 13C chemical shifts indicate a decrease in both the c and f 13C chemical shifts compared to the HDA moiety (36.8 ppmR33.1 ppm and 29.3 ppmR27.7 ppm respectively). In the case of the trans-3-hydroxydodec-5-enoic acid, an increase in these same chemical shifts is predicted (36.8 ppmR39.1 ppm and 29.3 ppmR33.7 ppm respectively). In the cis configuration, the van der Waals radii of the hydrogens of the two carbons flanking the double bond are overlapping due to their spatial proximity, as opposed to the trans configuration. This steric compression causes an additional shielding and thus upfield shift of the associated carbons, which is not present in the case of the trans configuration (for reference, see Breitmaier E., Voelter E. (1987) Carbon-13 NMR Spectroscopy: High-Resolution Methods and Applications in Organic Chemistry and Biochemistry, 3rd edition. Weinheim: VCH Verlagsgesellschaft mbH. 515 p.). Acknowledgments The authors wish to thank M. Ho¨fte and J. Xu (Laboratory of Phytopathology, Ghent University, Belgium) for assistance in testing xantholysin sensitivity of Xanthomonas oryzae strains. J. Goeman (Laboratory of Organic and Bioorganic Synthesis, Ghent University) is acknowledged for assistance in LC-MS and high resolution MS analysis. Table S1 Characterization of antibacterial activity of P. putida BW11M1. Homology of genes disrupted in BW11M1 Table S1 Characterization of antibacterial activity of P. putida BW11M1. Homology of genes disrupted in BW11M1 3. Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34: 1037–1062. (PDF) Therefore, this provides a significant indication for the lipid tail in xantholysin variant 2 to be in the cis-configuration. (PDF) Table S4 Analysis of bacterial susceptibility to P. putida BW11M1. List of non-Xanthomonas bacteria used as indicators. (PDF) Table S5 Antifungal activity of P. putida BW11M1 and xantholysin-deficient mutants. The effect on mycelial growth near a bacterial colony is indicated: (+) inhibition by wild type; (Q) or (2), reduced or no inhibition by xtl mutants compared to WT (as observed on PTA medium). For a particular indicator, the same phenotype was observed for the xtlA, xtlB, xtlC and xtlR mutants (CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively), collectively designated xtl mutants. The inhibitory patterns for selected fungal strains are shown in Fig. S5. (PDF) 7. Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GEM, et al. (2004) Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms. Mol Microbiol 51: 97–113. (PDF) The experi- mental values of the 3-hydroxydodec-5-enoic acid moiety compared to those of the HDA moiety (37.91 ppmR35.53 ppm and 29.60 ppmR27.28 ppm for the c and f 13C nuclei respectively) show that the 13C chemical shifts correspond significantly better to the case of the cis-configuration, indeed showing a decrease. Therefore, this provides a significant indication for the lipid tail in xantholysin variant 2 to be in the cis-configuration. Figure S17 NMR analysis of fatty acid moiety in xantholysin C. 1H and 13C chemical shift comparison between the lipid tail of xantholysin A and variant 2. (A) Experimental 1H (red) and 13C (blue) chemical shifts of the xantholysin 3- hydroxydecanoic acid moiety, compared to predicted 13C chemical shifts (green). (B) Experimental 1H (red) and 13C (blue) chemical shifts of the xantholysin variant 2 3-hydroxydodec-5- enoic acid moiety, compared to predicted 13C chemical shifts (green) assuming a cis-configuration. (C) Predicted 13C (green) chemical shifts of the xantholysin variant 2 3-hydroxydodec-5- enoic acid moiety assuming a trans-configuration. 13C chemical shift predictions were calculated using ChemNMR Pro 12.0 as implemented in the ChemDraw Ultra 12.0 software (PerkinElmer, Inc.). In the case of cis-3-hydroxydodec-5-enoic acid, the predicted 13C chemical shifts indicate a decrease in both the c and f 13C chemical shifts compared to the HDA moiety (36.8 ppmR33.1 ppm and 29.3 ppmR27.7 ppm respectively). In the case of the trans-3-hydroxydodec-5-enoic acid, an increase in these same chemical shifts is predicted (36.8 ppmR39.1 ppm and 29.3 ppmR33.7 ppm respectively). In the cis configuration, the van der Waals radii of the hydrogens of the two carbons flanking the double bond are overlapping due to their spatial proximity, as opposed to the trans configuration. This steric compression causes an additional shielding and thus upfield shift of the associated carbons, which is not present in the case of the trans configuration (for reference, see Breitmaier E., Voelter E. (1987) Carbon-13 NMR Spectroscopy: High-Resolution Methods and Applications in Organic Chemistry and Biochemistry, 3rd edition. Weinheim: VCH Verlagsgesellschaft mbH. 515 p.). The experi- mental values of the 3-hydroxydodec-5-enoic acid moiety compared to those of the HDA moiety (37.91 ppmR35.53 ppm and 29.60 ppmR27.28 ppm for the c and f 13C nuclei respectively) show that the 13C chemical shifts correspond significantly better to the case of the cis-configuration, indeed showing a decrease. 4. Raaijmakers JM, De Bruijn I, De Kock MJD (2006) Cyclic lipopeptide production by plant-associated Pseudomonas spp.: diversity, activity, biosynthesis, and regulation. Mol Plant Microbe Interact 19: 699–710. 2. 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https://openalex.org/W4231682318	https://hal.archives-ouvertes.fr/hal-03212570/file/Subbotina%2C%20boskovic%2C%20Diaz%2C%20Gery%2C%202021-24.pdf	French	null	Introduction	Cahiers du MIMMOC	2,021	cc-by	1,273	To cite this version: Galina Subbotina, Sanja Boskovic, Elvire Diaz, Catherine Géry. Introduction. Mémoire(s), iden- tité(s), marginalité(s) dans le monde occidental contemporain. Cahiers du MIMMOC, 2021, Figures de femmes dans les cultures européennes, Regards croisés France-Asie centrale : l’enseignement des langues étrangères à l’université (24), 10.4000/mimmoc.6921. hal-03212570 HAL Id: hal-03212570 https://hal.science/hal-03212570v1 Submitted on 12 Jul 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. Introduction Galina Subbotina, Sanja Boskovic, Elvire Diaz et Catherine Géry Éditeur Universit Université de Poitiers Ce document vous est offert par Université de Poitiers Ce document vous est offert par Université de Poitiers Référence électronique Référence électronique q Galina Subbotina, Sanja Boskovic, Elvire Diaz et Catherine Géry, « Introduction », Mémoire(s), identité(s), marginalité(s) dans le monde occidental contemporain [En ligne], 24 \| 2021, mis en ligne le 30 mars 2021, consulté le 21 juin 2021. URL : http://journals.openedition.org/mimmoc/6921 ; DOI : https://doi.org/ 10.4000/mimmoc.6921 Ce document a été généré automatiquement le 21 juin 2021. Mémoire(s), identité(s), marginalité(s) dans le monde occidental contemporain – Cahiers du MIMMOC est mis à disposition selon les termes de la licence Creative Commons Attribution 4.0 International. Introduction Introduction 1 1 Introduction Galina Subbotina, Sanja Boskovic, Elvire Diaz et Catherine Géry Galina Subbotina, Sanja Boskovic, Elvire Diaz et Catherine Géry 1 Le présent numéro des Cahiers du MIMMOC réunit les articles des participant.es de la Journée d’études Figures de femmes dans les cultures européennes : mémoires, identités, marginalités qui a eu lieu à l’Université de Poitiers les 3 et 4 octobre 2019. Cette rencontre, qui se voulait pluridisciplinaire, s’est donné pour but de refléter, d’une part, la multiculturalité et la multipolarité du monde contemporain, et d’autre part, la complexité de la place des femmes qui ne peut être décrite qu’à travers un ensemble de différentes disciplines. En évitant de simplifier ou de rendre invisibles les diversités, nous avons cherché à étudier des figures de femmes européennes, chacune se trouvant dans une situation unique, un contexte culturel particulier et se heurtant, dans ses quêtes identitaires, à des enjeux de pouvoir différents. Avec nos interventions consacrées à plusieurs pays de l’Europe de l’Ouest, de l’Europe médiane et de l’Europe de l’Est, nous nous sommes attachés à créer une mosaïque, un tableau multifacette afin de retracer plusieurs parcours de femmes qui, restant souvent à l’ombre ou en marge de la vie sociale et culturelle, parviennent, malgré tout, à faire entendre leur voix et à influencer le monde autour d’elles. 2 Dans cette publication, la problématique de marginalisation et d’invisibilisation des femmes fusionne avec deux autres questions qui se trouvent en corrélation sur le plan social et psychologique. Dans un premier groupe de textes, nous avons proposé d’étudier le problème de la mémoire sociale et de la présence des femmes dans les récits historiques et canoniques. L’article de Catherine Géry donne le ton à ces réflexions dans son analyse de l’œuvre de Maria Joukova, écrivaine russe du XIXe siècle, ainsi que des mécanismes d’effacement de son nom dans le canon littéraire russe. Elvire Diaz aborde, dans son étude, le rôle important des personnages féminins dans les romans dits « de la mémoire » au sein de la littérature espagnole post Transition démocratique (1975). A son tour, Sanja Boskovic, à travers des œuvres d'André Gide, de Thomas Mann et de Milorad Pavic, analyse la fonction de l'héritage mythique dans la constitution des figures de femmes dans leurs poétiques respectives. 3 Une place importante est occupée, dans cette publication, par les études consacrées à l’histoire des femmes de l’époque soviétique. Introduction Mémoire(s), identité(s), marginalité(s) dans le monde occidental contemporain, 24 \| 2021 Introduction Ainsi, Hélène Menegaldo se tourne vers la Mémoire(s), identité(s), marginalité(s) dans le monde occidental contemporain, 24 \| 2021 2 Introduction biographie d’Irène Kakhovskaïa, militante du Parti socialiste révolutionnaire de gauche (internationaliste), éradiqué par les bolcheviques dès 1918. Le nom de Kakhovskaïa, qui a passé l’essentiel de sa vie en prison sous le régime tsariste, puis soviétique, n’est pas entré dans les manuels d’histoire. L’activiste a partagé en cela le destin de plusieurs mouvements politiques du début du XXe siècle en Russie et en Ukraine. Le but de l’article d’Amine Lagoune est aussi de retracer l’histoire des femmes soviétiques sous des aspects effacés pour des raisons idéologiques. L’auteur de l’étude traite le rapatriement des ressortissantes soviétiques de France en URSS après la fin de la Seconde guerre mondiale. Le deuxième groupe de textes du présent numéro réunit des réflexions autour de la question de la subjectivité et de la quête identitaire. L’article de Susan Böhmisch fait le lien avec la problématique de la mémoire. Son étude vise à souligner l’impact souvent minimisé, voire oblitéré, des femmes sur l’histoire de l’art, et à montrer les pratiques culturelles et subjectives utilisées par les peintres Paula Modersohn-Becker, Gabriele Münter et Marianne von Werefkin afin de contourner de nombreux obstacles et de trouver leur place dans la vie artistique allemande du début du xxe siècle. Pour sa part, Ekaterina Belavina se focalise sur la question des identités littéraires et leur formation sous diverses influences, y compris étrangères : la chercheuse consacre son article à l’œuvre de Marceline Desbordes-Valmore et à sa réception en Russie par plusieurs poètes et poétesses des XIXe et XXe siècles. Galina Subbotina, de son côté, met en valeur le caractère novateur des textes autobiographiques féminins dans la littérature romantique russe, dans laquelle l’expression de soi est extrêmement difficile à cause de plusieurs interdits sociaux. L’aspect complexe de la mythologie autobiographique construite autour de l’image de la mère est étudié par Valentina Chepiga dans son article consacré à l’œuvre de Romain Gary. Quant à Françoise Defarges, elle analyse les textes littéraires de Lidia Zinovieva-Annibal, écrivaine russe du tournant des XIXe et XXe siècles, qui élabore plusieurs modèles originaux d’identité en transformant les représentations identitaires traditionnelles. La création de modèles de féminité capables d’impacter les spectatrices est au cœur de l’étude d’Andja Srebro qui s’intéresse à la place des personnages féminins dans le cinéma yougoslave de l’après-guerre. Mémoire(s), identité(s), marginalité(s) dans le monde occidental contemporain, 24 \| 2021 Introduction Malgré la diversité des situations nationales et historiques décrites dans ce numéro, il est possible de distinguer plusieurs points communs abordés et, en premier lieu, la minimisation du rôle des femmes ou l’effacement de leurs noms dans les « Grands récits » de la modernité (Jean-François Lyotard). Toutes ces études réunies montrent que la réintégration des femmes dans les métarécits est nécessaire, car elle permet de transformer de manière substantielle les visions normatives en changeant, par exemple, les dates d’apparition « des premiers » textes ou des « premières » œuvres, régulièrement « oubliés » quand il s’agit de la production féminine. Plusieurs articles du recueil mettent en évidence l’actuelle émergence de la « question féminine » à côté d’autres récits marginalisés comme, par exemple, les histoires de mouvements sociaux et culturels ne correspondant pas aux tendances dominantes. Cette émergence permet, entre autres, de relativiser l’importance des récits canoniques se présentant souvent comme « irremplaçables » ou « sans alternative » et de mettre en lumière le fonctionnement de « systèmes de vérité » (Michel Foucault) qui déterminent la place centrale ou périphérique des phénomènes sociaux et qui résultent des luttes, des conflits, des négociations complexes entre instances de pouvoirs divers.
https://openalex.org/W3194474982	https://pubs.rsc.org/en/content/articlepdf/2021/ma/d1ma00608h	English	null	A novel turn-on fluorescent sensor for cyanide ions based on the charge transfer transition of phenothiazine/indolium compounds	Materials advances	2,021	cc-by	6,559	a Forensic Science Laboratory, Kyoto Prefectural Police H.Q., 85-3, Yabunouchi-cho, Kamigyo-ku, Kyoto, 602-8550, Japan. E-mail: morikawa.fsl.kyoto@gmail.com b Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka, 577-8502, Japan. E-mail: k-nishi@phar.kindai.ac.jp c Antiaging Center, Kindai University, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka, 577-8502, Japan † Electronic supplementary information (ESI) available: Detailed experimental section, photography, UV-vis measurements, 1H NMR analyses and other addi- tional data. See DOI: 10.1039/d1ma00608h Materials Advances View Article Online View Journal \| View Issue Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5:48:54 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Yasuhiro Morikawa,a Miku Hirabara,b Keiji Nishiwaki, *b Shigeo Suzukibc and Isao Nakanishibc A new fluorescent sensor combining phenothiazine and indolium, which reacts specifically with cyanide ions with a large Stokes shift and a good fluorescence quantum yield, was prepared. When CN was added to an ethanol solution containing the synthesized sensor molecules, the solution color changed from purple to colorless, and fluorescence was emitted under 270 nm light irradiation. The mechanism of this luminescence, investigated via proton nuclear magnetic resonance, electrospray ionization mass spectrometry, and computational analysis, was defined as follows: the sensor undergoes nucleophilic addition of cyanide to the carbon of C = N+ in its indolium moiety, which limits the intramolecular charge transfer, resulting in a colorless transition and blue fluorescence. The specificity of the proposed sensor for cyanide has been confirmed through tests with 14 other anions (F, Cl, Br, I, ClO3 , ClO4 , NO2 , NO3 , SO3 2, SO4 2, S2O3 2, S2, N3 , and SCN) and the detection limit is 0.02 mM in the fluorescence spectrum. Changes in this sensor color can be detected by the naked eye and fluorescence emission can be induced via black light irradiation. Received 14th July 2021, Accepted 17th August 2021 DOI: 10.1039/d1ma00608h rsc.li/materials-advances Received 14th July 2021, Accepted 17th August 2021 rsc.li/materials-advances A novel turn-on fluorescent sensor for cyanide ions based on the charge transfer transition of phenothiazine/indolium compounds† Cite this: Mater. Adv., 2021, 2, 6104 Introduction advanced facilities, and excellent operation skills. Hence, sev- eral chemical sensors, including colorimetric sensors and fluorescent sensors that selectively react with CN, have been recently developed for the rapid detection of CN in the field.16 This approach has advantages such as shorter inspection time and lower cost, and allows simple and rapid on-site determina- tion and is extremely useful for practical applications. Many sensors have been reported and most of them are based on the nucleophilic addition of CN to their molecules17–20 or the coordination of cyano groups to metal reagents.16,21–23 The cyanide ion has extremely high toxicity but is relatively easy to obtain; it is used in the metal plating and plastics industries, and as a reagent in most laboratories.1 Given the concern about the health eﬀects of CN release into the environment, various technologies have been developed to detect CN in drinking water and similar, mainly in the environmental field. CN is also involved in serious crimes;2 for example, cyanide gas is produced in fires in addition to carbon monoxide.3 Therefore, CN detection is extremely important to determine the cause of death in homicides and fires. In this study, we attempted to develop a turn-on fluorescent sensor for CN, although fluorescence emission generally has a low detection limit; in other words, we designed a molecule that emits fluorescence when reacting with CN. There have been many recent reports of detection reagents based on the mechanism of quenching of fluorescence that should be emitted via intramolecular charge transfer (ICT) and emitting fluorescence by cutting oﬀthe charge transfer.24–26 In the molecular design, an electron-donating group and an electron-withdrawing group are connected by a carbon–carbon double bond to form a donor–p–acceptor system. Although various compounds are possible, we selected phenothiazine as the electron-donating group and an indolium salt as the electron-withdrawing group. A similar combination has been reported by El-Shishtawy et al. as a dimethine cyanine dye (PTZIS), a candidate for nonlinear optical materials.27 Thus, many instrumental analytical methods such as gas chromatography, mass spectrometry, liquid chromatography, and ion chromatography have been applied for the detection of CN;4–15 however, these methods require expensive equipment, © 2021 The Author(s). Published by the Royal Society of Chemistry 6104 \| Mater. Synthesis of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI) 1H-NMR (600 MHz, CDCl3) d 8.40 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 15.8 Hz, 1H), 7.73–7.42 (m, 6H), 7.16 (t, J = 7.3 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 6.97 (d, J = 6.4 Hz, 2H), 6.88 (d, J = 8.0 Hz, 1H), 4.38 (s, 3H), 3.89 (t, J = 6.7 Hz, 2H), 1.81 (s, 8H), 1.53–1.06 (m, 12H), 0.86 (d, J = 6.6 Hz, 3H). 13C-NMR (151 MHz, CDCl3) d 181.02, 153.45, 151.02, 142.57, 142.44, 141.61, 132.95, 130.03, 129.56, 129.14, 128.14, 127.67, 127.46, 124.37, 124.01, 123.17, 122.47, 116.23, 115.78, 114.15, 109.57, 51.90, 48.36, 36.88, 31.71, 29.20, 29.15, 27.22, 26.84, 26.77, 22.61, 14.10. IR (KBr, cm1) 2925, 2852, 1653, 1586, 1570, 1514, 1461. HRMS (ESI, positive mode, m/z): calculated for [C33H39N2S]+ 495.2828, found 495.2818. Scheme 1 shows the synthetic route of PI, which was modified from the method reported by El-Shishtawy et al.27 10-Octyl-10H-phenothiazine (1). Potassium hydroxide (2.08 g, 370 mmol) was added portionwise to a solution of Scheme 1 Synthesis of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)- 1,3,3-trimethyl-3H-indol-1-ium iodide (PI). Chemicals and instruments Phenothiazine and n-octyl bromide were purchased from Nacali Tesque. 1,2,3,3-Tetramethyl-3H-indolium iodide was provided by Santa Cruz Biotechnology, Inc. Unless otherwise noted, all the chemicals were obtained from commercial suppliers and used without further purification. Pure water was prepared with a Milli-Q system (Millipore Corporation). Fluorescence spectra and ultraviolet-visible light (UV-vis) absorption spectra were recorded on a FP-6200 (Jasco Corporation) and a UV-2700 spectrophotometer (Shimadzu Corporation), respectively. ESI-MS spectra were obtained using a micrOTOF II (Bruker Daltonics, Inc.) or an EXACTIVE mass spectrometer (Thermo Fisher Scientific). 1H-NMR spectra were measured with a JEOL JNM-AL400 (400 MHz) and a Bruker Avance (600 MHz) spectrometer in dimethyl sulfoxide (DMSO)- d6, with tetramethylsilane as an internal standard. Melting points were determined using a J-SCIENCE RFS-10 micro melt- ing point apparatus and not corrected. A Jasco FT/IR-460 Plus spectrophotometer was used to measure infrared (IR) spectra. (E)-2-(2-(10-Hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-tri- methyl-3H-indol-1-ium iodide (PI). A solution of 2 (195.4 mg, 0.576 mmol) and 1,2,3,3-tetramethyl-3H-indolium iodide (190.5 mg, 0.632 mmol) in dry ethanol (5.5 mL) was stirred at 60 1C for 10 h. After the solvent was removed by vacuuming, the residue was diluted with ethyl acetate, followed by the addition of n-hexane to the precipitate; the resulting precipitate was filtered oﬀto give the title compound (300 mg, 84% yield) as a dark blue solid and, then, was further purified via recrystalliza- tion from a methanol–diethyl ether solution for the analysis. (E)-2-(2-(10-Hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-tri- methyl-3H-indol-1-ium iodide (PI). A solution of 2 (195.4 mg, 0.576 mmol) and 1,2,3,3-tetramethyl-3H-indolium iodide (190.5 mg, 0.632 mmol) in dry ethanol (5.5 mL) was stirred at 60 1C for 10 h. After the solvent was removed by vacuuming, the residue was diluted with ethyl acetate, followed by the addition of n-hexane to the precipitate; the resulting precipitate was filtered oﬀto give the title compound (300 mg, 84% yield) as a dark blue solid and, then, was further purified via recrystalliza- tion from a methanol–diethyl ether solution for the analysis. Introduction Adv., 2021, 2, 6104–6111 6104 Materials Advances View Article Online View Article Online Paper Materials Advances 10H-phenothiazine (2.92 g, 14.6 mmol), 1-bromooctane (3.45 g, 17.8 mmol), and potassium iodide (40.3 mg, 0.24 mmol) in DMSO (50 mL). The reaction mixture was stirred at room temperature for 29 h and, then, poured into water (200 mL). The reaction mixture was extracted with chloroform. The combined chloroform layer was washed with saturated NH4Cl aqueous solution, dried over sodium sulfate, and concentrated in vacuo; the crude product was purified via silica gel flash column chromatography (hexane) to give 4.28 g, 94% yield as a colorless oil. The spectral data of 1 were in good agreement with the reported values.31 Phenothiazines emit fluorescence and have electron-donating properties,28–31 while many studies have reported the reaction of the indolium cation, which is a strong electron-withdrawing group, with CN.32–44 Therefore, we thought that a phenothia- zine/indolium compound could also be applied for CN detection. We synthesized a novel phenothiazine/indolium conjugated molecule (PI) as a CN sensing dye; its color and fluorescence changes in response to CN, as well as the selectivity to the target analyte, were also investigated. Then, its reaction mecha- nism was analyzed through proton nuclear magnetic resonance (1H-NMR), electrospray ionization mass spectrometry (ESI-MS), and computational analysis. Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5:48:54 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 10-Octyl-10H-phenothiazine-3-carbaldehyde (2). Phosphoryl chloride (2.74 mL, 29.3 mmol) was added dropwise at 0 1C to ice-cooled N,N-dimethylformamide (5 mL, 64.6 mmol) under a nitrogen atmosphere. The reaction mixture was warmed up to room temperature, stirred for 10 h, and then cooled down to 0 1C, followed by the addition of a solution of 1 (2.19 g, 7.02 mmol) in N,N-dimethylformamide (2.5 mL). The resulting mixture was heated to 80 1C, stirred for 18 h, and then poured into ice water; afterward, it was basified by adding saturated potassium carbonate aqueous solution and extracted with chloroform. The combined organic phase was dried over sodium sulfate and concentrated in vacuo. The crude product was purified via flash column chromatography (n-hexane/ethyl acetate = 9 : 1) to give the title compound (2.02 g, 85% yield) as a yellow oil. The NMR and IR spectral data of 2 were in good agreement with the reported values.31 UV-Vis and fluorescence measurements Fig. 1 compares the absorption and fluorescence spectra of PI and the fluorescent product in diﬀerent solvents. Therefore, PI was dissolved in various solvents, such as methanol, ethanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and chloroform; then, CN (2 equiv.) was added to these solutions, and the solution color change and fluorescence were analyzed. The excitation wavelength was 270 nm because it provided the maximum fluorescence when adding CN to PI in the ethanol solution, and the corresponding fluorescence wavelength was 483 nm, giving a larger Stokes shift (ESI,† Fig. S1). Fig. 2 also shows photographs of the color change of the solution and the fluorescence excited at 365 nm. The PI solution in chloroform showed blue color, while in the others it showed purple. El- Shishtawy et al. have observed the same phenomenon in PTZIS; they attributed this to the interaction of the iodide ion of the counter anion with the chlorine atoms in chloroform, which reduces the electron density of the iodide ion and destabilizes the ground state, enhancing the ICT characteristics and caus- ing a bathochromic shift.27 When adding CN, the color tone disappeared and all the solutions exhibited fluorescence with large Stokes shifts. The fluorescence intensity of the fluorescent product was weak in chloroform; we attributed this to the large bathochromic shift caused by ICT in that solvent. Among the tested solvents, dimethyl sulfoxide showed the strongest fluorescence intensity, but suﬃcient fluorescence intensity was observed in solvents other than chloroform also. In the case of chloroform, the dipole moment of the excited state of the fluorescent product was smaller due to the lower polarity of the solvent, and the maximum absorption wavelength has led to the bathochromic shift. Therefore, we decided to use etha- nol, which is more practical and safer, in the subsequent experiments. A quartz cuvette with an optical path length of 1 cm was used for all the measurements. The UV-vis spectra were collected in the 300–800 nm range. The fluorescence spectra were recorded between 300 and 700 nm under 270 nm light irradiation. Preparation of sample solutions for UV-vis and fluorescence measurements CN aqueous solution was added to 3 mL of 10 mM PI solutions and each spectrum was measured. For optimization of the solvents, 6 mL (2 equiv.) of a 10 mM CN aqueous solution, for the determination of the quantitative relationship of Scheme 1 Synthesis of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)- 1,3,3-trimethyl-3H-indol-1-ium iodide (PI). Mater. Adv., 2021, 2, 6104–6111 \| 6105 © 2021 The Author(s). Published by the Royal Society of Chemistry 6105 View Article Online Materials Advances Paper Paper product produced by adding CN to PI were performed at the B3LYP/6-31G level of theory by using the Gaussian 09 package.47 CN reacting with PI, 3–75 mL (0.1–2 equiv.) of a 1 mM CN aqueous solution and for selectivity to various anions, 6 mL of 10 mM aqueous anion solution were added. Each aqueous anion solution was prepared by dissolving 100 mmol of one among 15 inorganic salts (KCN, NaF, NaCl, NaBr, KI, KClO3, KClO4, NaNO2, NaNO3, Na2SO3, Na2SO4, Na2S2O3, Na2S, NaN3, and KSCN) in 10 mL of pure water. CN reacting with PI, 3–75 mL (0.1–2 equiv.) of a 1 mM CN aqueous solution and for selectivity to various anions, 6 mL of 10 mM aqueous anion solution were added. Each aqueous anion solution was prepared by dissolving 100 mmol of one among 15 inorganic salts (KCN, NaF, NaCl, NaBr, KI, KClO3, KClO4, NaNO2, NaNO3, Na2SO3, Na2SO4, Na2S2O3, Na2S, NaN3, and KSCN) in 10 mL of pure water. UV-Vis and fluorescence measurements The fluorescence quantum yield (F) was determined via a compara- tive method by using 15 mM quinine sulfate dihydrate (F = 0.55 in 0.1 M H2SO4, where F is the integrated area under the corrected emission spectrum) as the standard according to the following equation:45 Fx = Fst (Ast/Ax) (Fx/Fst) (Zx/Zst)2 where A is the absorbance at the excitation wavelength, Z is the refractive index of the solution, and the subscripts x and st denote the unknown material and the standard, respectively. Job’s plot measurement A 20 mM solution of PI was prepared by dissolving in EtOH. Similarly, a 20 mM solution of CN was prepared by dissolving it in a 4 : 1 (v/v) mixture of EtOH/H2O. Mixture solutions (3.0 mL) of PI and CN at 11 diﬀerent ratios from 0 : 3 to 3 : 0 (v/v) were prepared. After the solutions were allowed to stand for 10 min, the absorbance at 555 nm was measured at room temperature. The diﬀerence between the initial absorbance (A0) and the obtained absorbance value (A) was plotted against [PI]/ ([PI] + [CN]), and the complex formation ratio was estimated from the abscissa at the maximum absorbance. Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5:48:54 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5:48:54 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Computational analysis The structural optimization and computational calculations for the ground state of PI were conducted via density functional theory (DFT) at the B3LYP/6-311G level of theory using Spar- tan’10,46 while those for the excited states of the fluorescent Fig. 1 (a) Absorption and (b) fluorescence spectra (lex = 270 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI, 10 mM) in various solvents (3 mL) before and after adding CN. Fig. 1 (a) Absorption and (b) fluorescence spectra (lex = 270 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI, 10 mM) in various solvents (3 mL) before and after adding CN. © 2021 The Author(s). Published by the Royal Society of Chemistry © 2021 The Author(s). Published by the Royal Society of Chemistry 6106 \| Mater. Adv., 2021, 2, 6104–6111 Materials Advances View Article Online View Article Online Materials Advances Fig. 2 (a) Changes in the color and (b) the fluorescence excited by black light (l = 365 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI, 10 mM) in various solvents (3 mL), before and after adding CN. the slope of the calibration curve near the detection limit, was 0.29 mM by UV-vis and 0.02 mM by UV-vis fluorescence spectro- scopy. Although many fluorescent sensors for CN detection have been reported,48 PI showed relatively good sensitivity compared with these sensors (ESI,† Table S1). The CN concen- tration in human blood is toxic when it exceeds 19 mM,49,50 but the World Health Organization (WHO) has set the maximum concentration of CN in drinking water to be 1.9 mM.51 There- fore, PI could be applied in the environmental field or for forensic analysis because the LOD is much lower than these concentrations. The reaction kinetics between PI and CN were also inves- tigated. CN (20 mM, 2 equiv.) was added to a 10 mM PI solution and the change in the fluorescence intensity was measured over time (ESI,† Fig. S3(a)). The reaction started upon the CN addition and reached a steady state after 100 s. When ln[(Intt Intmax)], where Intmax is the emission intensity at the steady state and Intt is the intensity at time t, was plotted versus time, a linear fit was obtained, as shown in Fig. S3(b) (ESI†), demon- strating that the reaction follows pseudo-first-order kinetics. The rate constant of this reaction (Kobs), calculated via the above reaction equation, was 0.0307 s1 (ESI,† Fig. S3(b)). Changes in the absorption and fluorescence spectra of PI when adding CN The quantitative relation between PI and CN was studied via UV-vis and fluorescence spectroscopy analyses. The absorbance at 390 and 555 nm decreased with increasing CN concen- tration (Fig. 3(a)), while the fluorescence increased with it (Fig. 3(b)). The fluorescence intensity at 483 nm varied linearly with the CN concentration (R2 = 0.9945) (ESI,† Fig. S2). The limit of detection (LOD), calculated as 3.3s/a, where s is the standard deviation of the signal in the blank material and a is Fig. 3 (a) Absorption and (b) fluorescence spectra (lex = 270 nm) of 10 mM 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1- ium iodide when adding various concentrations of CN. The insets in (a) and (b) show the changes in the solution color and fluorescence, respec- tively, when adding CN. Computational analysis Then, the reaction rate was determined in the same way for CN and less than one equivalent of solutions containing 10, 7.5, and 5 mM of CN were added to a 10 mM solution of PI. The fluorescence intensity increased linearly up to 50 s and then slowly; after 20 minutes of measurement, no steady state was reached, but the fluorescence intensity of each solution was proportional to the respective CN concentration (ESI,† Fig. S4). Fig. 2 (a) Changes in the color and (b) the fluorescence excited by black light (l = 365 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI, 10 mM) in various solvents (3 mL), before and after adding CN. Changes in the absorption and fluorescence spectra of PI when adding CN Changes in the absorption and fluorescence spectra of PI when adding CN Mater. Adv., 2021, 2, 6104–6111 \| 6107 Selectivity of PI for diﬀerent anions To investigate the selectivity of PI for anions other than CN, 15 species of anions including CN were added to the PI solution and their behavior was observed. In addition, CN and other anions were added at the same time, and anti-interference experiments were also performed. As a result, the purple color disappeared and fluorescence was observed only when adding CN (Fig. 4). The absorbance intensity at 555 nm and the fluorescence intensity at 483 nm excited at 273 nm were also measured (Fig. 5, ESI,† Fig. S5 and S6). Among the other 14 anions, S2 induced a change in the solution color, which became lighter, and fluorescence emission, but the fluores- cence intensity was about 1/7 that obtained for CN. To investigate the reactivity of PI to S2, various S2 con- centrations (1–16 equiv.) were added to PI (ESI,† Fig. S7); the purple color disappeared with increasing S2 concentration and the absorption spectrum became constant at 4 equiv., but the fluorescence did not increase with it. The mass spec- trum of the reaction solution was measured, but only the peak of the cationic component of PI was observed, that is, the peak of the PI/S2 conjugated compound was not observed. These results indicate that S2 reacted with PI, but its reactivity is low. Besides, the estimated quantum yield of the reaction products was very low. Thus, PI could discriminate between CN and S2 and, in terms of fluorescence, it has specificity for CN. Fig. 3 (a) Absorption and (b) fluorescence spectra (lex = 270 nm) of 10 mM 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1- ium iodide when adding various concentrations of CN. The insets in (a) and (b) show the changes in the solution color and fluorescence, respec- tively, when adding CN. Mater. Adv., 2021, 2, 6104–6111 \| 6107 © 2021 The Author(s). Published by the Royal Society of Chemistry 6107 Fig. 4 (a) Changes in the color and (b) the fluorescence excited by black light (l = 365 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI) when adding 2 equiv. of diﬀerent anions. Fig. 5 (a) Changes in the absorbance at 555 nm and (b) fluorescence intensities of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H- indol-1-ium iodide (PI) at 483 nm at an excitation wavelength of 270 nm when adding 2 equiv. of diﬀerent anions. Materials Advances Paper View Article Online View Article Online View Article Online Fig. Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5 This article is licensed under a Creative Commons Attribution 3.0 Un Fig. 7 Electrospray ionization mass spectrum of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI) with CN (positive ion mode); the insets show the measured and theoretical values of PI and (E)-1,3,3-trimethyl-2-(2-(10-octyl-10H-phenothiazin-3- yl)vinyl)indoline-2-carbonitrile, respectively. Scheme 2 Plausible light-emitting mechanism of 2-(2-(10-hexyl-10H- phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI) with CN. corresponding to the protons on the (E)-olefin moiety (J = 16 Hz) shifted to 6.86 and 6.33 ppm (J = 16 Hz), respectively, and the signal of the methyl protons on the nitrogen atom shifted from 4.11 to 2.71 ppm. These results suggest that the chemical shifts of these hydrogens changed significantly because indolium was converted into a tertiary amine upon the CN addition and the olefin moiety conjugated to indolium ceased to be conjugated to the resulting tertiary amine. The binding mechanism between PI and CN was further confirmed via the MS measurements. The ESI-MS spectra of the PI/CN conjugated compound revealed the cationic component of PI (MW: 495.28) and [M + H]+ of the CNadduct of PI (cationic component) (MW: 522.29) (Fig. 7). Moreover, their isotopic ratio was consistent with the theoretical value. This result supports the nucleophilic addition of CN to the PI molecule, and the detection of the cationic component of PI as a fragment ion peak suggests that the cation is very stable. Scheme 2 Plausible light-emitting mechanism of 2-(2-(10-hexyl-10H- phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI) with CN. of PI is replaced with a methyl group to reduce the computa- tional cost. The higher occupied molecular orbital (HOMO) of PI was widely distributed from the phenothiazine ring to the C = N+ site of the indolium group (Fig. 8); this suggests that ICT is occurring, which may have resulted in the loss of fluores- cence. The gap between the HOMO and lower unoccupied molecular orbital (LUMO) was 2.10 eV (590 nm), which corre- lates well with the experimental data. The large coeﬃcient on the carbon of the C = N+ moiety in the LUMO also explains the nucleophilic addition of CN to it. The HOMO–LUMO gaps of both the enantiomers of PICN were also calculated and they showed the same value (3.92 eV), higher than that of PI (Fig. 9). This increase in the energy gap implies the breakage of the p-conjugation between the phenothiazine and indolium moi- eties and it implies that the ICT process has been stopped. Selectivity of PI for diﬀerent anions 4 (a) Changes in the color and (b) the fluorescence excited by black light (l = 365 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI) when adding 2 equiv. of diﬀerent anions. Materials Advances Paper ed Licence. Materials Advances Paper Fig. 4 (a) Changes in the color and (b) the fluorescence excited by black light (l = 365 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI) when adding 2 equiv. of diﬀerent anions. ed Licence. Fig. 4 (a) Changes in the color and (b) the fluorescence excited by black light (l = 365 nm) of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3- trimethyl-3H-indol-1-ium iodide (PI) when adding 2 equiv. of diﬀerent anions. Fig. 5 (a) Changes in the absorbance at 555 nm and (b) fluorescence intensities of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H- indol-1-ium iodide (PI) at 483 nm at an excitation wavelength of 270 nm when adding 2 equiv. of diﬀerent anions. hed on 18 August 2021. Downloaded on 10/24/2024 5:4 licensed under a Creative Commons Attribution 3.0 Un Fig. 5 (a) Changes in the absorbance at 555 nm and (b) fluorescence intensities of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H- indol-1-ium iodide (PI) at 483 nm at an excitation wavelength of 270 nm when adding 2 equiv. of diﬀerent anions. Identification of the fluorescent compound and sensing mechanism Job’s plots. A 1H-NMR titration study in DMSO-d6 was con- ducted to acquire an insight into the proposed reaction path- way of CN and PI (Fig. 6). A CN solution was added at 0.25– 1.25 equiv. to PI solution incrementally. According to the amount of CN added, the signals at 8.30 and 7.52 ppm To determine the structure of the PI/CN conjugated compound, we performed 1H-NMR and MS measurements after the CN addition to PI. The ratios of the reactants were determined via Fig. 6 Proton nuclear magnetic resonance spectra of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide before and after the CN addition, in various concentrations, in dimethyl sulfoxide-d6. Fig. 6 Proton nuclear magnetic resonance spectra of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide before and after the CN addition, in various concentrations, in dimethyl sulfoxide-d6. © 2021 The Author(s). Published by the Royal Society of Chemistry © 2021 The Author(s). Published by the Royal Society of Chemistry © 2021 The Author(s). Published by the Royal Society of Chemistry 6108 \| Mater. Adv., 2021, 2, 6104–6111 View Article Online Paper Paper Materials Advances Fig. Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5 This article is licensed under a Creative Commons Attribution 3.0 Un Moreover, to confirm the generation constants of PI and CN, we analyzed the generation ratio via Job’s plot analysis. The max- imum value was observed at 0.5, which means that PI and CN react at a ratio of 1:1 (ESI,† Fig. S8). From these results, we concluded that the reaction between PI and CN is a CN addition to the C = N+ of the indolium ring, yielding (E)-1,3,3-trimethyl-2-(2- (10-octyl-10H-phenothiazin-3-yl)vinyl)indoline-2-carbonitrile (PICN). As reported for many CN sensors containing indolium, the CN addition to the carbon of the double bond of indolium breaks the conjugation between phenothiazine and indolium, linked by a double bond, inhibiting the ICT from the electron-donating (phe- nothiazine) to the electron-withdrawing group (indolium) (Scheme 2). As a result, quenching in the visible part and fluores- cence emission should occur. The Stokes shift and F of PICN were, respectively, 118 nm and 0.60 relative to quinine (F = 0.55 in 0.1 M H2SO4) (ESI,† Table S2). Therefore, PI reacts with the cyanide ion with a large Stokes shift and a good F. Time-dependent DFT calculations for the excited state of PICN were also performed to verify the emission. The fluores- cence wavelength of both PICN enantiomers was 481 nm, which Fig. 8 Frontier molecular orbitals and their energies of 2-(2-(10-hexyl- 10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI). Selectivity of PI for diﬀerent anions 7 Electrospray ionization mass spectrum of 2-(2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI) with CN (positive ion mode); the insets show the measured and theoretical values of PI and (E)-1,3,3-trimethyl-2-(2-(10-octyl-10H-phenothiazin-3- yl)vinyl)indoline-2-carbonitrile, respectively. Open Access Article. Published on 18 August 2021. Downloaded on 10/24/2024 5:48:54 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Computational analysis The frontier orbitals were obtained through DFT calculations for a compound where the n-octyl group on the phenothiazine Fig. 8 Frontier molecular orbitals and their energies of 2-(2-(10-hexyl- 10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI). Fig. 8 Frontier molecular orbitals and their energies of 2-(2-(10-hexyl- 10H-phenothiazin-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (PI). © 2021 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2021, 2, 6104–6111 \| 6109 View Article Online Paper Materials Advances Fig. 9 Frontier molecular orbitals of (E)-1,3,3-trimethyl-2-(2-(10-octyl- 10H-phenothiazin-3-yl)vinyl)indoline-2-carbonitrile (PICN) and their energies. Conclusions 10 P. Luo, Y. Yu, D. Wu, X. Li, C. Dai, X. Chen, G. Li and Y. Wu, Anal. Methods, 2019, 11, 2983–2990. We designed and synthesized a new fluorescent chemical sensor, PI, and investigated its response to CN. PI showed a clear color change and fluorescence when adding CN, as well as suﬃcient sensitivity with a LOD of 0.02 mM, which is below the maximum value established by the WHO and the toxic CN concentration in human blood. Among 15 anions, PI reacted only with CN; that is, it emitted fluorescence specifically in response to CN and its fluorescence intensity increased line- arly when adding 0–1.3 equiv. CN. 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https://openalex.org/W4287348482	https://zenodo.org/record/5540344/files/11.pdf	English	null	The use of social media in the creation of personal learning environment during the #studyfromhome period	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	7,203	Journal of Education and Learning (EduLearn) Vol. 15, No. 1, February 2021, pp. 78~87 ISSN: 2089-9823 DOI: 10.11591/edulearn.v15i1.17581 Journal of Education and Learning (EduLearn) Vol. 15, No. 1, February 2021, pp. 78~87 ISSN: 2089-9823 DOI: 10.11591/edulearn.v15i1.17581  78  78 78 ABSTRACT Due to the Covid-19 outbreak in Indonesia the Government urged students and lecturers to conduct the learning process from home through an online system called #studyfromhome. The architecture undergraduate students in this study were millennial students who used digital technology on a daily basis. This study was conducted to determine the role of social media, as well as millennial students’ preferences and feedback on the use of social media as learning tools to create a personal learning environment (PLE). Questionnaires were distributed online to students of Class of 2017 and 2018, out of whom 115 respondents provided their feedback. The results of the study showed that the students of the Architecture Study Program at Udayana University in Bali were fond of using social media especially audio-visual applications for learning activities. In addition, very positive feedback was also provided in terms of knowledge sharing and creativity, acquisition of information, and submission of assignments. Social media were considered more student-friendly. This condition was relevant to the characteristics of the millennial students who were independent learners, and facilitated the creation of PLE. Dealing with the new approach, the students hoped that social media could be used in a better manner as architecture learning platforms. Architecture learning Digital technology Millennial students Personal Learning Environment Social media This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author: Ni Ketut Agusintadewi Master of Architecture Program Universitas Udayana Jalan PB Sudirman, Denpasar, Bali 80234, Indonesia Email: nkadewi@unud.ac.id Corresponding Author: Ni Ketut Agusintadewi Master of Architecture Program Universitas Udayana Jalan PB Sudirman, Denpasar, Bali 80234, Indonesia Email: nkadewi@unud.ac.id The use of social media in the creation of personal learning environment during the #studyfromhome period Ni Ketut Agusintadewi1, Ni Made Mitha Mahastuti2, Kadek Agus Surya Darma3, Anak Agung Ngurah Aritama4 1 1Master of Architecture Program, Universitas Udayana, Indonesia 2,3,4Architecture Study Program, Universitas Udayana, Indonesia 1Master of Architecture Program, Universitas Udayana, Indonesia 2,3,4Architecture Study Program, Universitas Udayana, Indonesia 1. INTRODUCTION When the Covid-19 pandemic reached Indonesia, the government made the #studyfromhome appeal to all schools and universities throughout Indonesia. The appeal, which was enforced on 16 March 2020, was expected to flatten the curve of the rapid transmission of Covid-19. Hence, online learning became the only option to carry out the learning process safely from home. Various social media, such as WhatsApp, YouTube, Zoom, Webex, Facebook, Instagram, Line, Telegram, and others, began to be used effectively as learning tools. Digital technology and its derivatives are rapidly developing to bridge the gap between space and time in distance learning activities between learners and teachers. Digital technology in teaching and learning activities at universities has long been widely used in the 21st century. This technology-based education is highly relevant to the characteristics of the younger generation whose learning styles are different from previous generations. The students of this generation are classified as a ‘millennial’ generation, or ‘Gen Y’, who were born between 1980 and 2000 [1, 2]. The Journal homepage: http://journal.uad.ac.id/index.php/EduLearn J Edu & Learn 79  ISSN: 2089-9823 millennial generation is considered a ‘digital native’ generation because they have been familiar with technology since their childhood [3]. This generation is tech-savvy, and they have a high dependency on digital technology [4]. millennial generation is considered a ‘digital native’ generation because they have been familiar with technology since their childhood [3]. This generation is tech-savvy, and they have a high dependency on digital technology [4]. The development in student literacy attached with advanced technology has emerged the increase of digital technology in education [5]. The rapid development of digital technology presents its own challenges for teaching staff in the learning process. Students’ learning styles also change, resulting in differences in values, perspectives, and approaches between generations. The millennial generation expect to be involved in their learning and being active learners by digital technology [5], so the lectures must have digital learning platform, include the smart phones, laptop, computers and the internet. Besides digital devices, learning process in architecture education not only needs average computer programs, but it is supported by specific software, namely AutoCAD and Sketchup. Interestingly, students have been simultaneously motivated by using digital technology, particularly using modelling and Computer-Aided Design (CAD) technology in the process of design [6]. 1. INTRODUCTION Changes in the meaning of learning in the mindset of millennial learners in the higher education environment will result in changes in mindset and action patterns in the climate and learning environment to answer the challenges of the times [7]. The challenges faced by higher education today are student learning styles and lack of enthusiasm of lecturers in utilizing technology as a learning tool [8]. In addition to that, there is a wide gap between senior lecturers and millennial students [9]. The incompatibility between lecturer teaching styles and student learning styles may cause decreased student interest in learning, which may lead to a lack of self-confidence [10]. To understand the characteristics of millennial students, there are four main characteristics of millennial generation students: Innovation; Investigation; Immediacy; and Authentication and Trust [10]. Table 1 illustrates these four characteristics with the additional characteristic of ‘Social Consciousness’ as referenced from a number of other studies. Table 1. Characteristics of millennial students Main characteristics Black (2010) [11] Noor (2016) [10] Suh and Hargis (2016) [12] Innovation  The use of technology at a higher level  Aesthetic aspect in the presentation of course materials Investigation  Curiosity, discovery, and exploration  Curiosity and exploration Immediacy  Demanding, impatient, and less focused  Constantly keep up with the development of cyberspace information Table 1. Characteristics of millennial students Main characteristics Black (2010) [11] Noor (2016) [10] Suh and Hargis (2016) [12] Innovation  The use of technology at a higher level  Aesthetic aspect in the presentation of course materials Investigation  Curiosity, discovery, and exploration  Curiosity and exploration Immediacy  Demanding, impatient, and less focused  Constantly keep up with the development of cyberspace information Authentication and trust  Digital native Social consciousness  More open and confident  Tend to work in groups and show teamwork  Have high tolerance and respect for differences  Have networks outside the geographical area  Share the values that are instilled by their family and community On the other hand, this generation gap also provides interesting opportunities for lecturers to engage students in different ways. Lecturers can get higher class participation and longer student attention, and build interaction with students if they make class presentations using technology, such as online videos and Power Point slides [13]. The use of social media in the creation of personal learning environment during … (Ni Ketut Agusintadew 1. INTRODUCTION Another study also found that students would be more interested in class assignments related to and requiring digital technology [14]. Digital technology on smartphones can also be used to find out feedback from students quickly [1, 13]. Mobile learning by utilizing gadgets is a very effective choice because students commonly use mobile devices [15]. Connections with digital technology can make the learning process more efficient and more accessible to students [4], thus creating an affinity space that is suitable for the place where the learning activities take place [16]. The affinity space offers a new approach in learning methods facilitated by various technologies, such as social media including WhatsApp, YouTube, Telegram, Twitter, Facebook and Instagram. Social media are defined as various network technology tools that emphasize on the social aspect of the Internet as a channel for communication, collaboration, and creative expression [17]. Millennial students use social media to share information, create social networks outside their geographical reach, seek new knowledge, share academic activities, and other things they learn in college  80 ISSN: 2089-9823 [18, 19]. They are “active content co-producers” [18] and have become part of the international virtual community [18]. Due to their extensive network and unlimited content, social media can be used for academic discussion activities outside classrooms [20]. The use of social media can facilitate the creation of a Personal Learning Environment (PLE), and thus, social media have become a system that can help millennial students control and manage their own learning [18, 20], as shown in Table 2. [18, 19]. They are “active content co-producers” [18] and have become part of the international virtual community [18]. Due to their extensive network and unlimited content, social media can be used for academic discussion activities outside classrooms [20]. The use of social media can facilitate the creation of a Personal Learning Environment (PLE), and thus, social media have become a system that can help millennial students control and manage their own learning [18, 20], as shown in Table 2. Table 2. How social media facilitate the creation of PLE Previous researchers PLE creation method Blaschke (2014) [21] 1. Promote self-potential and develop abilities for the creation of PLE. 2. More self-directed learners. 3. The uniqueness of the learning activities that affects meta-cognition skills: a. Construct new knowledge b. Formulate learning contents c. Understand the learning process individually Dabbagh and Kitsantas (2012) [18] 1. 1. INTRODUCTION Share knowledge with everyone in social networks 2. Participate in building collective knowledge 3. Manage the process of building individual knowledge Table 2. How social media facilitate the creation of PLE Previous researchers PLE creation method In addition to facilitating a learning environment outside the classroom, social media also offer the millennial generation diverse benefits for learning, as described in Table 3. Table 3. The benefits of social media for millennial learning Previous researchers Benefits of social media in learning Blaschke (2014) [21] 1. Social media are used to support learning activities and the learning process itself. Coleman (2013) [22] 1. Enable students to have freedom to have connections and collaborate off campus to gain practical knowledge in the world of work Dabbagh and Kitsantas (2012) [18] 1. Enable students to search for information and share information/knowledge. 2. Allow students to participate in knowledge sharing collectively and manage individual knowledge. Hussain (2012) [19] 1. Facilitate exchange of academic activities, sharing of learning experiences, and development of social networks. 2. Facilitate collaboration and accessibility to develop virtual communities across countries. Rahimi, van den Berg and Veen (2013) [23] 1. Enable students to have the opportunity to manage the learning environment or become an independent learner. Greenhow and Robelia (2009) [24] 1. Fulfill the social function of learning which penetrates the spaces of activities for the learner. Kurkela (2011) [25] 1. Social media related to competency development are more important for independent learners. Through social media, the visual aspect of millennial learning can be optimized. The millennial generation are visual learners because they prefer reflective learning styles and visual learning compared to other generations [26, 27]. This includes learning by reading graphs and diagrams, thinking in pictures, and making illustrations [10]. Millennial students absorb visual information better than information provided using conventional approaches in the form of texts and lectures [28]. They are also more adept at visual presentation techniques [11], and that is why the millennial generation is classified as visual learners [29]. In response to the development of the 21st century’s higher education, the #studyfromhome period at Udayana University was an early milestone in the use of digital technology as an intensive architectural learning tool. Social media are the right choice and are relevant with millennial students’ learning styles. In the past, social media were not used optimally as learning platforms by architecture lecturers and students. 1. INTRODUCTION Therefore, it was necessary to conduct this research to evaluate the teaching-learning process during the Covid-19 pandemic. This study analyzes the feedback provided by millennial students in connection with their learning experiences on the effectiveness of the use of social media as architectural learning tools during the #studyfromhome period. This research revealed the role of social media, millennial students’ preferences and feedback on the use social media as learning tools for discussions and assignment consultations, as well as presentations and submission of assignments, for both theoretical and architectural studio courses. 2.1. Research design This research was designed with a mix-method approach. In the classroom research, the student quantitative data were collected by distributing the Likert scale questionnaires, while the qualitative data J Edu & Learn, Vol. 15, No. 1, February 2021: 78 – 87 J Edu & Learn  81 ISSN: 2089-9823 were collected through structured interviews and a number of open-ended questions on the questionnaire [30]. The students were given an opportunity to describe their learning experiences during the #studyfromhome period to gain more insights on their opinions, hopes, and aspirations. The questionnaires were developed to identify the respondents’ preferences and the role of social media, while open-ended questions allowed for feedback from the respondents, especially on their attitudes towards the use of social media in architectural learning. were collected through structured interviews and a number of open-ended questions on the questionnaire [30]. The students were given an opportunity to describe their learning experiences during the #studyfromhome period to gain more insights on their opinions, hopes, and aspirations. The questionnaires were developed to identify the respondents’ preferences and the role of social media, while open-ended questions allowed for feedback from the respondents, especially on their attitudes towards the use of social media in architectural learning. The questionnaires in the form of Google Forms were distributed to the respondents through a link sent via WhatsApp. The data collection process was conducted for two weeks starting from 15 June 2020 or during the implementation of the Final Semester Examination after all lecture learning activities for the Even Semester of the Academic Year 2019-2020 had been completed. Students had enough time to experience and reflect on their learning activities on the whole, which included online lectures, preparation, assignment completion, and post-assignment submission. Interviews were conducted via voice calls. Both types of data were analyzed statistically and interpreted qualitatively. 2.2. Questionnaires The questions for the questionnaires were prepared based on the purpose of the study. The questionnaire was prepared by referring to a number of previous studies. The themes and question variables are presented in Table 4. Table 4. Linkages among themes, variables and survey activities Themes Variables Survey activities Student learning characteristics  Innovation  Investigation  Immediacy  Authentication and trust  Social consciousness Online questionnaires Creation of student PLE through social media  Promote self-potential  Develop the ability to create PLE (self-directed learners)  Construct new knowledge through learning content  Participate in building collective knowledge by sharing knowledge through social networks Learning activities  Read information presented in tables, schemes, and pictures  Use search engines to search for information  Look for inspiration from existing architectural works through online simulations  Read textbooks (theories, concepts, nonfiction content)  Interact with computer simulations  Listen to lectures through video recordings and video conferencing  Conduct discussion and present individual and group assignments to lecturers through social media  Online learning evaluation system Preferences in the use of social media  Social media tools that are suitable for theory class learning  Social media tools that are suitable for architectural studio activities The benefits of social media in learning  Support learning activities and processes  Provide ease in interacting with other architecture students or using other learning resources off campus globally  Search for and share information faster and more extensively  Support collective knowledge building and individual knowledge management (independent learners)  Promote competencies and learning outcomes to the wider communities (Source: Formulated from the relevant studies 2020) Table 4. Linkages among themes, variables and survey activities Themes Variables 2.3. Respondents The respondents of this study were Architecture Study Program students of Class of 2017 and 2018. The students of both classes were born in the year between 1998 and 2000 or aged 20 to 22 years, and can be categorized as Millennial Generation. Class of 2019 was not chosen as respondents because they had insufficient learning experience in architecture compared to the previous classes, which made their critical thinking and problem-solving skills less developed. Class of 2016 was not selected either because most of them were taking an Architecture Studio Final Assignment. The number of respondents who were willing to fill out the questionnaire was 115 students. 3. RESULTS AND DISCUSSION 3.1. Students’ preferences in the use of social media for learning activities 3.1. Students’ preferences in the use of social media for learning activities nces in the use of social media for learning activitie The data collected from the questionnaire showed that of the 115 respondents who provided feedback, 49% were women and 51% were men, with the majority or 58% aged 20 years, as shown in Figure 1. The data on the respondents’ age showed that 85% of the respondents were in the category of the millennial generation with an age range of 20-22 years or a birth year of 1998–2000. (a) (b) Figure 1. Distribution of respondents providing feedback (a) Gender distribution; (b) Age range Female Students (49%) Male Students (51%) 19 year-old (15%) 20 year-old (58%) 21 year-old (24%) 22 year-old (3%) (b) 19 year-old (15%) 20 year-old (58%) 21 year-old (24%) 22 year-old (3%) (a) Female Students (49%) Male Students (51%) 22 year-old (3%) Female Students (49%) 20 year-old (58%) (b) Figure 1. Distribution of respondents providing feedback (a) Gender distribution; (b) Age range Figure 2 shows that 54% of the respondents gave a score of 5 (Love it), 32% gave a score of 4 (Like it), and 14% gave a score of 3 (Neutral). None of the respondents gave a score of 4 (Dislike it) or 5 (Dislike it very strongly). This finding shows that millennial students have a high preference (around 86%) for using social media in learning activities and like this approach. This high preference reflects the characteristics of the millennial generation that are highly dependent on digital technology [3, 4]. Figure 2. Millennial students’ preference in using social media Figure 2. Millennial students’ preference in using social media As many as 66.4% of the millennial students said that social media was beneficial for them, especially in searching for and sharing information faster and more extensively, supporting learning activities, and enabling students’ freedom to interact with other learning resources outside the campus, as shown in Table 5. Table 5. The benefits of social media for millennial learners Benefits of social media Percentage 1. Support learning activities 21.6 2. Provide freedom to interact with other architecture students or other learning resources outside the campus 16.6 3. Enable faster and more extensive information searches and sharing 28.2 4. Support collective knowledge building and individual knowledge management (independent learners) 8.3 5. 2.4. Research constraints Because the research subjects are students of architecture, the findings of this study are only releva r architecture learning. In the future, similar research can be conducted on Generation Z. The use of social media in the creation of personal learning environment during … (Ni Ketut Agusintadew ISSN: 2089-9823  82 J Edu & Learn, Vol. 15, No. 1, February 2021: 78 – 87 1. Support learning activities 3.2. The role of social media in the creation of personal learning environment for millennial stude As commonly known about the millennial generation, Table 6 describes that 31.4% of the respondents stated that social media were able to encourage students’ abilities to be self-directed learners and 22.6% acknowledged that the use of social media was able to help construct new knowledge relevant to the learning content. This condition was triggered by the social media’s capacity to share knowledge and information from individuals to a wider social network (19.4%), thus allowing the respondents to participate in building collective knowledge (15.1%). This atmosphere is very much needed for the creation of PLE among millennial students as independent learners in controlling and managing their learning activities [18, 21]. Table 6. The role of social media in the creation of PLE The role of social media Percentage 1. Promote students’ self-potential 7.0 2. Develop students’ abilities as self-directed learners 31.4 3. Enable students to construct new knowledge through learning content 22,6 4. Enable students to participate in building collective knowledge 15.1 5. Enable students to motivate themselves and share individual knowledge within social networks 19.4 6. Other: Act as a window to information 4.5 Table 6. The role of social media in the creation of PLE The role of social media 6. Other: Act as a window to information Table 7 shows four learning activities that are most frequently done by millennial students, namely interacting with computer simulations (18.3%), seeking inspiration from architectural works through social media (16.4%), using search engines to search for information (15.5 %), and listening to lectures through video recordings and video conferencing (15.4%). This finding shows that millennial students are visual learners, and for this reason conventional learning activities, such as reading textbooks, become less interesting [29]. In the case of architecture students, visual learning is more relevant and attractive to millennial students. Table 7. Most frequently done learning activities Learning activities Percentage 1. Read information presented in tables, schemes, and pictures 7.1 2. Use search engines to search for information 15.5 3. Look for inspiration from existing architectural works through social media 16.4 4. Read textbooks (theories, concepts, nonfictions) 5.3 5. Interact with computer simulations 18.3 6. Listen to lectures through video recordings and video conferencing 15.4 7. Consult on assignments with lecturers through the online system 8.1 8. Present individual and group assignments to lecturers through social media 6.4 9. Discuss architectural studio assignments within the assignment cluster through social media 7.4 10. 3. RESULTS AND DISCUSSION Facilitate social network building in the field of architecture globally 11.7 6. Promote students’ competencies and learning outcomes to the wider communities 13.6 Table 5. The benefits of social media for millennial learners Benefits of social media Percentage 1. Support learning activities 21.6 2. Provide freedom to interact with other architecture students or other learning resources outside the campus 16.6 3. Enable faster and more extensive information searches and sharing 28.2 4. Support collective knowledge building and individual knowledge management (independent learners) 8.3 5. Facilitate social network building in the field of architecture globally 11.7 6. Promote students’ competencies and learning outcomes to the wider communities 13.6 Table 5. The benefits of social media for millennial learners Benefits of social media Percentag J Edu & Learn, Vol. 15, No. 1, February 2021: 78 – 87 83 ISSN: 2089-9823 J Edu & Learn  Of all the applications used, WhatsApp, Line, and Instagram are the most widely chosen tools for architectural learning. The reasons cited varied widely, but most respondents agreed that these social media applications were commonly used, and made it easy for them to interact and share information in the form of text, graphics, audio and video, both between individuals and within groups. In addition, most students acknowledged that they had been using social media since junior high school. This is consistent with the characteristics of the millennial generation who are very familiar with digital technology, even from an early age [4]. 3.2. The role of social media in the creation of personal learning environment for millennial stude Online learning evaluation system 7.2 Table 7. Most frequently done learning activities 2. Use search engines to search for information The social media that best suit the learning needs of theoretical courses are those that provide chat and voice call facilities (WhatsApp, Line), data transfer facilities (WhatsApp, Line), video call facilities (WhatsApp), and video conferencing facilities (Zoom, Webex). Meanwhile, the applications most suitable for architectural studio learning needs are those through which students can share presentation data with fellow group participants, such as Zoom and Webex (for two-way communication), as well as Instagram and YouTube (for one-way communication). The use of social media in the creation of personal learning environment during … (Ni Ketut Agusintadew  84 ISSN: 2089-9823 3.3. Students’ feedback on the use of social media for learning activities After familiarizing themselves with social media, most of the students stated that they experienced an increase in Social Consciousness. As many as 22.6% of the respondents acknowledged having high tolerance and respect for differences, 22.3% stated that they had a social network outside their geographical area, and 17.6% acknowledged that they were able to share the values instilled by their family and community. This is shown in Table 8. These findings confirm the characteristics of the millennial generation who tend to be more open, flexible, and willing to share with a wider network, without being limited by space and time [10]. Table 8. The benefits of social media in increasing social consciousness Social consciousness Percentage 1. Become more open and self-confident 13.1 2. Tend to be in groups rather than being alone 8.4 3. Have high tolerance and respect for differences 22.6 4. Have social networks outside their geographical area 22.3 5. Tend to collaborate with others rather than working alone 7.6 6. Increase their inner spirituality 6.2 7. Share the values that are instilled by their family and community 17.6 8. Other: Tend to be individualistic 2.2 Table 8. The benefits of social media in increasing social consciousness Social consciousness Percentage 1. Become more open and self-confident 2. Tend to be in groups rather than being alone 3. Have high tolerance and respect for differences 8. Other: Tend to be individualistic Through the Word Cloud application, the frequency of words used in the respondents’ additional comments can be generated as shown in Figure 3. J Edu & Learn, Vol. 15, No. 1, February 2021: 78 – 87 J Edu & Learn from the internet that they are inclined to look for shortcuts without seriously considering neither the reliability of the information they get nor the ethicality of their conduct [31]. from the internet that they are inclined to look for shortcuts without seriously considering neither the reliability of the information they get nor the ethicality of their conduct [31]. In addition, the student feedback on the acquisition of knowledge and understanding supports the idea that the use of social media can help in the creation of student PLE [18, 21]. Students become better at understanding course material when interacting with assignments online, so that they experience cognitive processes, visual understanding, and constructive feedback from others. This is consistent with the characteristics of millennial students as visual learners who learn more efficiently through online applications [10]. The use of social media as platforms for collecting assignments is considered to be more student- friendly because it saves more money and time. This approach using social media is also convenient for students because they can learn in their own affinity space [16], anytime and anywhere they have access to online networks. Social media have become part of their daily lives, so this approach can be a supportive method in architecture learning for big classes with more than 20 students. Nevertheless, using social media as a learning platform should be effectively blended with class lectures and presentations for small classes. Digital learning, therefore, must be inevitably applied and developed for millennial students in higher education, as the Covid-19 health protocol suggested. With the right and convenient method, lecturers can spark the interest of the 21st century’s students, so that students’ intentions and efforts to learn can emerge naturally. Table 9. Selected feedback of the respondents Reasons Quantity of feedback Selected feedback Sharing of knowledge and creativity 27  I can share information to the public on social networks  Instagram is the platform most suitable for students to find design inspirations in completing architectural studio assignments  Social media are very easy to access and students can share the outcome of their assignments to their classmates.  Students can share their best architectural works on their networks on Instagram. New method of teaching 23  It is fun and easily accessible by anyone, so that anyone can learn from a different space, time, scenario, and perspective. J Edu & Learn  It is a new method at the Architecture Study Program of Udayana University and, therefore, lecturers and students are challenged to be more creative.  My friend overseas is also doing e-learning; it feels like I am studying abroad.  I prefer that evaluations are carried out through social media platforms to written tests. Acquisition and understanding of information 15  For me, this is an easy way to review assignments, so it will be easier to remember the lecture material.  It increases my understanding of the course material. This way of teaching should be maintained.  It’s easier for students to do learning activities because they are already familiar with digital technology. Exploration of ideas for architecture students will be easier to do because information from all over the world can be obtained easily. Ease of submitting assignments 12  It’s cheaper, no cost is needed to print assignments.  It’s more student-friendly, easy to access and use, more exciting.  It combines technology and education.  It’s environmentally friendly, paperless.  Using technology and the Internet makes it easier to complete assignments compared to using conventional methods. Intensity of social media use by the millennial generation 12  The use of social media is very suitable because almost every student has an account, not only for sharing photos, but also for sharing information.  I like this approach because it’s a combination of innovation and the latest trends. Millennials should have a social media account and use this platform to connect with others.  It’s the most appropriate way to contact students through social media applications that are commonly used by them, wherever they are. Table 9. Selected feedback of the respondents Table 9. Selected feedback of the respondents Reasons Quantity of feedback Selected feedb  I like this approach because it’s a combination of innovation and the latest trends. Millennials should have a social media account and use this platform to connect with others. p  It’s the most appropriate way to contact students through social media applications that are commonly used by them, wherever they are. The use of social media in the creation of personal learning environment during … (Ni Ketut Agusintadew 3.2. The role of social media in the creation of personal learning environment for millennial stude Based on the Word Cloud, the terms most frequently mentioned by the students were learning, audio-visual media, information sharing, new approach, fun, challenging, and interesting. In general, the frequency of these words reflects positive feedback from millennial students about their experiences using social media as platforms for architectural learning. Figure 3. Frequency of word usage Figure 3. Frequency of word usage Student feedback can be grouped into various preferences for the use of social media in architectural learning activities. Table 9 lists significant comments from each of these reason groups. This finding is consistent with the characteristics of millennial students, technological dependence, and the ways in which social media help learning activities. Appreciation of student knowledge and sharing of creativity through the collection of assignments on social media are in accordance with the role of students as content creators or influencers to members of their online networks [18, 19]. Some students acknowledged that they appreciated the positive feedback from their online followers for the creative content they had shared. They were also enthusiastic about the idea of using social media as a new and interesting approach in education. This further confirms their characteristics as innovative and inquisitive digital-native generation, fast learners [10]. On the other hand, having access to digital devices and internet almost all the time has also led digital learners to always determine for quick answers rather than longer problem-solving. Students may come to be in countless sets of information, but they have been able to sort the information obtained into useful data. This relevant to the characteristics of millennial students is used to getting instant information on almost anything J Edu & Learn, Vol. 15, No. 1, February 2021: 78 – 87 85  J Edu & Learn ISSN: 2089-9823 J Edu & Learn REFERENCES H. Raslie, S. Pit, and S. Ting, "Millennials’ expectations of life at the university and the workplace: A malaysia perspective," International Journal of Education, vol. 8, no. 3, pp. 71-84, 2016. [2] T.J. Smith and T. 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The use of social media in architectural learning is currently very beneficial for lecturers and millennial students, as long as the learning activities are adjusted to the learning outcomes.  86 ISSN: 2089-9823 Feedback from students also motivates lecturers to use social media in learning activities more intensively. Although quite a number of students think that online lectures reduce their interaction in the real world, most students recognize that the use of social media is very exciting and makes it easier for them to learn. Some students also prefer to maintain their privacy on social networks, so that lecturers need to encourage them to have a public account for academic and professional purposes. Having a public account is very useful and can encourage the creation of a PLE in which students become self-directed learners. Currently, many educational institutions and industries at the local, national and international levels already have accounts with which they share content on social networks. y This research is a preliminary study and serves to provide input for classroom/studio action research. Further research needs to be conducted to determine the effectiveness of using digital technology or social media in architectural learning activities after the Covid-19 pandemic. In addition, similar research can also be carried out on younger class of architecture students who were born after the year 2000, who are called Generation Z. Do Gen Y and Gen Z have different learning styles? What is their feedback on the use of digital technology in learning activities? Are there any differences in the effectiveness social media use in learning activities for theoretical courses and studio courses? These are a number of issues that need to be answered in future research. BIOGRAPHIES OF AUTHORS Ni Ketut Agusintadewi is an associate professor of architecture at the Master of Architecture Program, Faculty of Engineering, Universitas Udayana. She also works at the Architecture Study Program in the same faculty with the subject taught: Architecture Studio 1 and 2, Real Estate, Design and Project Management, and supervising for Internship Program and Final Project. Her concern is on various issues of methods and strategic approach for architecture education and learning, both theoretical issues and practical studios. Since joining the undergraduate program, she has been actively involved in many kinds of support for curriculum and institution development. Ni Ketut Agusintadewi is an associate professor of architecture at the Master of Architecture Program, Faculty of Engineering, Universitas Udayana. She also works at the Architecture Study Program in the same faculty with the subject taught: Architecture Studio 1 and 2, Real Estate, Design and Project Management, and supervising for Internship Program and Final Project. Her concern is on various issues of methods and strategic approach for architecture education and learning, both theoretical issues and practical studios. Since joining the undergraduate program, she has been actively involved in many kinds of support for curriculum and institution development. Ni Made Mitha Mahastuti is a lecturer at the Architecture Study Program, Faculty of Engineering, Universitas Udayana; and takes a place in teaching teams: Architecture Studio 1 and 2, Building Sciences and Utility 1 and 2, Introduction to Architecture, Indonesian Architecture, Basic Principles for Spatial Design, and Material Technology. She encourages undergraduate students to use social media as an architectural learning platform, especially during the Covid-19 pandemic, and spread it on digital learning system integrated within the university e-learning network. Kadek Agus Surya Darma is a lecturer at the Architecture Study Program, Faculty of Engineering, Universitas Udayana, with subjects: Architecture Studio 1 and 2, Form and Aesthetic Studio, Architectural Design Methods, Principles for Urban Design, Cost Budget Planning in Architecture, Site Planning, Building Structure, Design and Project Management; and supervising for Internship Program. He also enthusiastically participates within strategic methods of effectiveness in architectural learning process for today and future needs. Using digital technology as a learning platform is one of his interests. Anak Agung Ngurah Aritama is a lecturer at the Architecture Study Program, Faculty of Engineering, Universitas Udayana. REFERENCES Fesol, et al., "Learning style approaches for Gen Y: An assessment conducted in a Malaysian Technical University," Pertanika Journal of Social Science & Humanities, vol. 24, no. 4, pp. 1335-1347, 2016. [27] S. F. A. Fesol, et al., "Learning style approaches for Gen Y: An assessment conducted in a Malaysian Te University " Pertanika Journal of Social Science & Humanities vol 24 no 4 pp 1335-1347 2016 [27] S. F. A. Fesol, et al., "Learning style approaches for Gen Y: An assessment conducted in a Malays University," Pertanika Journal of Social Science & Humanities, vol. 24, no. 4, pp. 1335-1347, 2016. y f pp [28] T. K Hoffman, T. Franks, and B. Edson, "Cultural awareness training: Preparing new in student," Basic Communication Course Annual, vol. 27, no. 1, pp. 10-19, 2015. [28] T. K Hoffman, T. Franks, and B. Edson, "Cultural awareness training: Preparing new instructors for the millennial student," Basic Communication Course Annual, vol. 27, no. 1, pp. 10-19, 2015. [29] J. Kriegel, Differences in Learning Preferences by Generational Cohort: Implications for Instructional Des Corporate Web-based Learning (Doctoral dissertation), Philadelphia, PA: Drexel University, 2013. [30] K. Liza, E. Andriyanti, "Digital literacy scale of English pre-service teachers and their perceived readiness toward the application of digital technologies," Journal of Education and Learning (EduLearn), vol. 14, no. 1, pp. 74-79, 2020. [31] M.A.M. Mohzan and H.A. Zubir, "Teaching the millennials: Implications on today's classrooms," Uni Journal of Educational Research, vol. 7, no. 9, pp. 186-191, 2019. REFERENCES Hanif, Asrowi, Sunardi, "Access to and perception of using mobile technologies in the classroom: The potential and challenges of implementing mobile learning," Journal of Education and Learning (EduLearn), vol. 12, no. 4, pp. 644-650, 2018. pp [16] J. D. Machin-Mastromatteo, "Participatory action research in the age of social media: literacies, affinity spaces and learning," New Library World, 113(11/12), pp. 571-585, 2012. [17] N. Dabbagh and R. Reo, "Back to the future: Tracing the roots and learning affordances of social software," in M.J.W. Lee & C. McLoughlin (Eds.), Web 2.0-based e-learning: Applying social informatics for tertiary teaching, pp. 1-20, 2011. pp [18] N. Dabbagh and A. Kitsantas, "Personal learning environments, social media, and self-regulated learning: a natural formula for connecting formal and informal learning," Internet and Higher Education, vol. 15, no. 1, pp. 3-8, 2012. [19] I. Hussain, "A study to evaluate the social media trends among university students," Procedia-Social and Behavioral Sciences, 2012, vol. 64, pp. 639-645. 0] S. Falahah and D. Rosmala, "Study of social networking usage in higher education environment," Procedia - Soc and Behavioral Sciences, 2012, vol. 67, pp. 156-166. [21] L. M. Blaschke, "Using social media to engage and develop the online learner in self-determined learning," Research in Learning Technology, vol. 22, pp. 1-23, 2014. g gy pp [22] V. Coleman, "Social media as a primary source: A coming age," EDUCAUSE Review, 2013. [Online] Available: https://er.educause.edu/articles/2013/12/social-media-as-aprimary-source-a-coming-of-age. J Edu & Learn, Vol. 15, No. 1, February 2021: 78 – 87  87 J Edu & Learn ISSN: 2089-9823 [23] E. Rahimi, J. van den Berg, and W. Veen, "Investigating teachers’ perceptions about the educational benefits of Web 2.0 personal learning environments," eLearning Papers, No. 35, 2013. [Online] Available: www.openeducationeuropa.eu/en/elearning_papers. p p g_p p [24] C. Greenhow dan E. Robelia, "Old communication, new literacies; social network sites as social learning resources," Journal of Computer-Mediated Communication, vol. 14, no. 4, pp. 1130-11161, 2009. [25] L. Kurkela, "Systematic approach to learning paradigms and the use of social media in higher educ International Journal of Emerging Technologies in Learning (iJET), vol. 6, pp. 14-20, 2011. f g g g g pp [26] J. Shepherd, A Causal-Comparative Study of Generational Differences in Learning Style Preferences among Adult Learners in the United States (Doctoral dissertation), La Verne, California: University of La Verne, 2017. ( ) y [27] S. F. A. BIOGRAPHIES OF AUTHORS He teaches several courses: Architecture Studio 3 and 4, Architecture Drawing 1 and 2, Engineering Drawing, Building Design Concept, Site Planning, Introduction to Human Settlements, and Architecture Visualization. Digital architecture is one of his concern that has motivated in elaborating digital learning platform in architecture education at the study program. The digital learning platform is integrated within the Universitas Udayana e-learning system: OASE or Online Academic Service E- Learning. use of social media in the creation of personal learning environment during … (Ni Ketut Agusintadewi)
https://openalex.org/W2587688735	https://ijpam.eu/contents/2016-111-1/10/10.pdf	English	null	RESPONSIVE USER INTERFACE FOR PEOPLE WITH ASD	International Journal of Pure and Applied Mathematics	2,016	cc-by	3,557	PA ijpam.eu PA ijpam.eu ISSN: 1311-8080 (printed version); ISSN: 1314-3395 (on-line version) url: http://www.ijpam.eu doi: 10.12732/ijpam.v111i1.10 RESPONSIVE USER INTERFACE FOR PEOPLE WITH ASD Nikolay Pavlov1, Kliment Mirchev2, Teodora Gardjeva3, Dilyana Krushkova4, Asen Rahnev5 § 1,3,4Plovdiv University 236, Bulgaria, Blvd., Plovdiv 4000, BULGARIA 29N Kuklensko shose, Blvd., Plovdiv 4000, Bulgaria 411-ti Avgust, Str., Plovdiv 4000, BULGARIA Abstract: This paper describes the design and implementation of the user interface of Open Book, a reading assistive tool for people with autism spectrum disorders (ASD). The ﬁndings in existing research played a vital role in UI implementation. Key screens of the user interface with the main features and functionality of the system are presented. The conducted evaluation by users with ASD and their caregivers clearly proves the usability and beneﬁts of the innovative software product. Key Words: responsive user interface, accessible user interface, users with autism spectrum disorders, project ﬁrst, open book 1. Introduction This paper describes the design and implementation of the user interface of Open Book. Open Book is a distributed software system employing various natural language processing components to convert documents for people with (Autistic Spectrum Disorders) ASD in a format easier to read and understand. Open Book is developed as a part of project FIRST (A Flexible Interactive Received: August 11, 2016 Revised: October 23, 2016 Published: December 6, 2016 © 2016 Academic Publications, Ltd. url: www.acadpubl.eu §C d h Received: August 11, 2016 Revised: October 23, 2016 Published: December 6, 2016 © 2016 Academic Publications, Ltd. url: www.acadpubl.eu © 2016 Academic Publications, Ltd. url: www.acadpubl.eu Published: December 6, 2016 §Correspondence author 106 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev Reading Support Tool) partially funded by the European Commission under the Seventh Framework Programme (FP7-2007-2013) for Research and Tech- nological Development under grant agreement # 287607. The FIRST project starts from the need to improve the life of people with ASD by enabling them to read any written documents and thus improving their social inclusion and interaction. People with ASD are approximately 3.3 million across Europe according to Autism Speaks research (see [2]). People with ASD may experience diﬃculties comprehending complex instructions while reading, getting misled by ﬁgurative language or the use of rare words or, simply, get distracted by secondary points touched upon in a document (see [1]). Using innovative language technology to simplify documents, the created software product Open Book helps ASD users convert a standard document into a personalized version which is easier for them to read and understand. 3. Approach to Design Open Book is not just being developed for people who have ASD, it is being developed with people who have ASD (see [4]). The team developing the tool was working with a large group of ASD users across three countries within the whole project duration in order to ensure that the tool will meet their diverse requirements and to evaluate the tool’s eﬀectiveness in improving reading comprehension. The ASD users’ engagement encompassed the whole project development and aimed to organize the desired assistive elements in a clear structure for the work packages involved in the development of the tool based on user re- quirements and to deﬁne in more precisely some important aspects for the development of the tool. Clinicians from three diﬀerent countries also took part in designing of the tool in order to create the most suitable user interface for people with ASD. The engagement of accessibility specialists played an important role in designing the UI. The result is intuitive, accessible, well-structured and responsive interface, suitable for both desktop and mobile devices. The UI is created according to the ﬁndings and recommendations in the paper User Interface for People with Autism Spectrum Disorders (see [3]). The research extracted guidelines for the look and feel of the interface desired by the ASD users. The ﬁrst prototype of the software, presented in this article, is created on the basis of this information. RESPONSIVE USER INTERFACE... 107 • Communication with the caregiver - if user still has problems with the document content after simpliﬁcation, he can send notiﬁcation to his carer sharing his problems and requirements for a particular document; • Preferences and selection of tools - user can select the default theme, font, create labels for the library organization and choose the most useful tools for his personal toolbar; 2. Primary Features of Open Book A quick review of the Open Book’s ﬁrst prototype basic features will help comprehend better the user interface of the tool, presented later on in this paper in section Design Implementation. This is the ﬁrst version of the tool and therefore the feature set is subject to further development and change. The system provides the following functions to users with ASD The system provides the following functions to users with ASD: • Simpliﬁcation - the text entered by user is automatically converted to a simpler and convenient format. If user still ﬁnds some word or expressions diﬃcult, he can ask the system for explanation, additional information or some images; • Personalization - user can adjust style, font and format of the simpliﬁed text according to his personal preferences and requirements. The system provides a set of specially designed by the clinical partners color themes which make the document reads better. However, user can also create his own color themes which best fulﬁll his special needs; • Improvement of readability - for user’s convenience the system provides features like highlighter and magniﬁer notes which help user to go less eﬀortlessly through the whole text or leave his thoughts and comments by using notes; • Better organization of documents - user can arrange his documents as he prefers - divide them into folders by using labels, mark them as ready or favorite, search and ﬁnd easily the desired document and others; RESPONSIVE USER INTERFACE... 4. Interface Features The main features of the user interface are: The main features of the user interface are: • Accessibility this design approach provides optimal view user experience easy reading and navigation with minimal eﬀorts for resizing and scrolling. 108 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev Figure 1: Demonstration of Open Book’s responsive design Figure 1: Demonstration of Open Book’s responsive design The buttons are kept as large as possible which enables easy navigation and work with the system (see [5]). The buttons are kept as large as possible which enables easy navigation and work with the system (see [5]). • Responsive user interface to support for devices with various form-factors - having in mind the increasing demands for tablets and mobile phones, UI is supported for a wide range of devices from desktop computer monitors to mobile devices. The web site supports a minimum of 800x600 desktop resolution, although we recommend using at least 1024x768. The support for mobile devices is maintained at an optimal quality at 480px and above screen width. • Personalization - font size, style and color, background theme and menu tools can be adjusted according to user’s personal needs and preferences. 109 RESPONSIVE USER INTERFACE... Figure 2: Support for desktop and mobile devices Figure 2: Support for desktop and mobile devices 5. Design Implementation Based on the ﬁndings and conclusions made in the previous article, the ﬁrst prototype of the Open Book software system implemented all components described. Its main goals are simple and clear design with minimum visual distractors, intuitive navigation, combination of pictures and words, simple graphics, clear fonts, large buttons, mild colors etc. which can be seen in the following examples. 6. Sign Up Long and large ﬁelds where user can enter his personal information are amongst the main requirements for a registration form for users with disabilities. Only most basic information (name, e-mail and password) is required here to complete the registration successfully. Big spaces are provided between the text ﬁelds, so each ﬁeld is clearly separated from the rest. Linear layout is used for the registration form in order to keep user’s attention focused on a single text column. Only the picture, which is not a requirement for a successful registration, can be found on the right side of the page. 110 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev Figure 3: Sign Up Screenshot Figure 3: Sign Up Screenshot 7. Simpliﬁed Document The simpliﬁcation page has simple and clear design which contains mini- mum visual distractors. Soft, mild colors and simple graphics are used for the presentation of the main options. The reading area occupies signiﬁcant space in this page presenting the simpliﬁed text to the user in in a large text box clearly separated from the rest. Researches show that ASD users get distracted by more than one column of text, so original and simpliﬁed text are disposed in diﬀerent tabs avoiding the option to stay next to each other in a single page and the need of sideways scrolling. This type of interface allows users to quickly focus their attention to the text area and adapt the presentation of document to their personal preferences. A single toolbar is provided in the simpliﬁcation page as a part of user requirements. The toolbar consists of big and clear buttons with both picture and text going next to each other - helping people with ASD understand better the meaning of the command. For the options like Bold, Underline and Picture Word-like icons are used leading the users to something common. • Theme - color themes are created after research about the color schemes that are best perceived by people with ASD. Themes correspond to the so called ”colour ﬁlters” used widely by persons with autism, Asperger syndrome, dyslexia and other reading disorders to focus their attention 111 RESPONSIVE USER INTERFACE... Figure 4: Simpliﬁed Document Screenshot Figure 4: Simpliﬁed Document Screenshot on the reading space. The most important feature here is the contrast between font and background. Colored ﬁlters have been found to reduce visual distortion of text in children with ASD, too (see [6]); • Font size - the system provides 6 diﬀerent sizes of font letters bigger than 12 pt due to the fact that ASD users prefer bigger letters than usual; • Underline, Bold and Highlight are three desired options taken from Word that people with ASD often use to emphasize information that they con- sider important or problematic; • Magnify option - this option helps user focus on particular sentence. Peo- ple with ASD can then go through the whole text sentence by sentence taking attention on each one separately; • Notes - users with ASD like leaving some notes whatever they do, remind- ing them of something important or just helping them share thoughts and feelings. 7. Simpliﬁed Document Open Book provides this functionality to the users enabling them write, save, view and delete notes in the simpliﬁed document. Trying to avoid pop-ups and other visual distractors the following UI behaviour was adopted: all messages returned by the interface and ad- ditional operations, related to the toolbar features appear in a textbox Trying to avoid pop-ups and other visual distractors the following UI behaviour was adopted: all messages returned by the interface and ad- ditional operations, related to the toolbar features appear in a textbox 112 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev Figure 5: Demonstration of magnify option Figure 5: Demonstration of magnify option under the main reading area. This allows user to receive all information requested meanwhile continues his work with the document. • Explain word and explain with picture - explanation of diﬃcult concepts with pictures and deﬁnitions is essential for improvement of reading com- prehension. Researches show that visual cues stimulate better the brain activity and help users get the meaning faster and easier. • Explain word and explain with picture - explanation of diﬃcult concepts with pictures and deﬁnitions is essential for improvement of reading com- prehension. Researches show that visual cues stimulate better the brain activity and help users get the meaning faster and easier. 8. Technologies Used 8.1. HTML5/CSS3/JS 8.1. HTML5/CSS3/JS The application deploys various recently developed and pushed by the web community CSS3 technologies such as the new Level 3 Selectors like: a) Selectors Level 3 - Pseudo-elements ::after, ::before; Pseudo-classes :last- child, :not; a) Selectors Level 3 - Pseudo-elements ::after, ::before; Pseudo-classes :last- child, :not; b) Media Queries; c) CSS Backgrounds and Borders Module Level 3 - background-repeat, border- radius; c) CSS Backgrounds and Borders Module Level 3 - background-repeat, border- radius; d) CSS Fonts Module Level 3; 113 RESPONSIVE USER INTERFACE... Figure 6: Demonstration of Explain with picture option Figure 6: Demonstration of Explain with picture option d) Chosen - simple to use library that creates and enriches the native HTML select element producing rich dropdowns and menus. 8.3. Responsive User Interface Responsive User Interface (UI) is implemented using the @media-query css technology. This technology depends on declaring css classes and rules in dif- ferent screen width ranges. Rules in those ranges overwrite rules from other classes and are considered high priority. The more commonly used width ranges are listed below: a) @media only screen and (min-width:600px) and (max-width:960px) - rules in this section apply when the screen width is between 600 pixels and 960 pixels; a) @media only screen and (min-width:600px) and (max-width:960px) - rules in this section apply when the screen width is between 600 pixels and 960 pixels; b) @media only screen and (max-width:600px) - rules in this section apply when the screen width is under 600 pixels; 8.2. JavaScript JavaScript is used extensively throughout the application mostly leveraging third-party libraries that bring to the table extremely useful tools to develop dynamic user interface, data exchange between the front-end ecosystem and the web application server as well as simply prettifying and stylizing libraries that add extra touch and feel to the whole application. The more prominent of those are: a) jQuery - this is a very profound and powerful library which supplies a whole plethora of functionalities for traversing, selecting and modifying the DOM. It also gives the powerful AJAX wrapper for executing asynchronous JavaScript-initiated XML requests towards a remote web application or ser- vice simply by passing a JavaScript object to the jQuery.ajax function. b) KnockoutJS - a powerful and simple to use JavaScript library for HTML binding. It uses Declarative Bindings which are mapped to a JavaScript ob- ject called ”the View Model”. It deploys automatic UI refresh and Depen- dency tracking which is extremely useful for developing business model-rich 114 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev Figure 7: Initiating an ajax call Figure 8: Declarative Knockout binding Fi 7 I iti ti j ll Figure 7: Initiating an ajax call Figure 7: Initiating an ajax call g g j Figure 8: Declarative Knockout binding Figure 8: Declarative Knockout binding dynamic JavaScript applications. Its declarative bindings are applied using the data-bind attribute, which maps an object or a variable of the View Model to the HTML element. dynamic JavaScript applications. Its declarative bindings are applied using the data-bind attribute, which maps an object or a variable of the View Model to the HTML element. c) MomentJS - powerful library for working with Date and Time in the JavaScript environment. The native JavaScript Date() object proves insuﬃcient and cumbersome to work with. The MomentJS library introduces a Date-wrapping object that provides a rich API for working with Date and Time d) Chosen - simple to use library that creates and enriches the native HTML select element producing rich dropdowns and menus. d) Chosen - simple to use library that creates and enriches the native HTML select element producing rich dropdowns and menus. 115 RESPONSIVE USER INTERFACE... Figure 9: Using and instantiating momentJS Figure 9: Using and instantiating momentJS 8.5. Browser support Open Book’s user interface is supported on the browsers, listed below: a) Internet Explorer 9+ b) Google Chrome 20+ b) Google Chrome 20+ c) Mozilla Firefox 10+ c) Mozilla Firefox 10+ d) Safari 5+ d) Safari 5+ 8.4. NET Framework NET Framework is used extensively both for back-end web request handling, serving HTML, ﬁles and data and for the Data Access layer. The OBS Web Application uses MVC 4 framework for handling web requests and the WCF framework for establishing connection with services. a) MVC 4 - the Web Application uses the Routing and Request handling of the MVC 4 framework which is very convenient and powerful for web appli- cations of all types. The views used for the diﬀerent pages are written using a) MVC 4 - the Web Application uses the Routing and Request handling of the MVC 4 framework which is very convenient and powerful for web appli- cations of all types. The views used for the diﬀerent pages are written using 116 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev Figure 10: Sample media queries Figure 10: Sample media queries Figure 10: Sample media queries Figure 11: MVC 4 Razor View Engine Figure 11: MVC 4 Razor View Engine the Razor View Engine which is very powerful for constructing sophisticated HTML constructions using a C# Model object. the Razor View Engine which is very powerful for constructing sophisticated HTML constructions using a C# Model object. the Razor View Engine which is very powerful for constructing sophisticated HTML constructions using a C# Model object. b) WCF - the OBS Web Application uses the WCF (Windows Communication Foundation) for consumption of the web services. The WCF client exposes a set of methods that are consumed by the web application with the pur- pose of receiving and sending document or user related data for storage and processing. The calls are made using a wrapping method call: 117 RESPONSIVE USER INTERFACE... Figure 12: WCF Service call wrapper Figure 12: WCF Service call wrapper 9. Testing and Evaluation Results Detailed clinical evaluation was conducted to assess eﬀectiveness of the Open Book software in reading comprehension of adults and children with high functioning autism. 294 people with ASD took part in the evaluation. The results clearly show that the majority of ASD users prefer simpliﬁed by Open Book texts than the original ones. When all participants (aside from controls) 118 N. Pavlov, K. Mirchev, T. Gardjeva, D. Krushkova, A. Rahnev were combined together, the simpliﬁed scores were 1.2 units higher than the original scores which deﬁnitely proves that the Open Book software improves reading comprehension (see [7]). Furthermore, feedback from caregivers of ASD people clearly proves that users ﬁnd documents converted with Open Book much more interesting and engaging than before. Children spend time reading texts on topics that they will normally refuse to read. That is because they consider the Open Book documents like entertainment and playing games. Their attention is taken by the interface features, the pictures presented for complicated words and personalization settings like change of fonts, themes, etc. for much longer period than the child’s normal attention span. 10. Conclusion Relying on guidelines described in the article ”User Interface for People with Autism Spectrum Disorders” (see [3]) the user interface of the Open Book Tool was designed and implemented. Up to now, the software have been tested and evaluated by more than 300 people. It has been used in practice for text simpliﬁcation and improvement of reading comprehension by people with ASD and clinicians from three diﬀerent countries. The beta version of the system can be found on the address http:// openbooktool.net/. The oﬃcial release of Open Book is planned for the end of 2014. RESPONSIVE USER INTERFACE... RESPONSIVE USER INTERFACE... 119 Acknowledgements The project leading to this research product is partially funded by the Euro- pean Commission under the Seventh Framework Programme (FP7-2007-2013) for Research and Technological Development under grant agreement # 287607 and by the IT15-FMIIT-004 project of the Scientiﬁc Fund of the University of Plovdiv ”Paisii Hilendarski”, Bulgaria. This paper reﬂects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein. We would like thank all the partners from the consortium of the FIRST project, and especially the clinical partners from Deletrea, Spain for their ex- tensive research, and Parallel World, Bulgaria for their invaluable contributions during the implementation of the user interface. 11. Future Development Results of the FIRST project can be used to beneﬁt other user groups except the people with ASD. Minor modiﬁcations of the system would be appropriate to adjust its settings to the preferences of other user groups like: • People with reading impairments like dyslexia; • Students in early grades; • Foreign language learners; References [1] Robert Sears, The Autism Book: What Every Parent Needs to Know About Early Detec- tion, Treatment, Recovery and Prevention, 2009. [2] Autism Speaks, http://www.autismspeaks.org [3] N. Pavlov, User interface for people with autism spectrum disorders, Journal of Software Engineering and Applications, 7, No. 2 (2014), Article ID: 43152. [3] N. Pavlov, User interface for people with autism spectrum disorders, Journal of Software Engineering and Applications, 7, No. 2 (2014), Article ID: 43152. [4] Newsletter, Second Edition, http://www.ﬁrst-asd.eu. [4] Newsletter, Second Edition, http://www.ﬁrst-asd.eu. [5] UI Design Guidelines for Responsive Design, http://tympanus.net/codrops/2013/01/21/ui- design-guidelines-for-responsive-design/ [6] A.K. Ludlow, E. Taylor-Whiﬀen, A.J. Wilkins, Coloured ﬁlters enhance the visual percep- tion of social cues in children with autism spectrum disorders, ISRN Neurol., 2012:298098 (2012), doi: 10.5402/2012/298098. [7] A. Cerga-Pashoja, V. Jordanova, LNFT, D7.6: Clinical Evaluation, http://ﬁrst- asd.eu/?q=FIRST documents. 120 120
https://openalex.org/W4312037862	https://www.repository.cam.ac.uk/bitstream/1810/343799/3/Humanists%20and%20scholastics%20in%20early%20sixteenth%20century%20Paris%20new%20sources%20from%20the%20Faculty%20of%20Theology.pdf	English	null	Humanists and scholastics in early sixteenth-century Paris: new sources from the Faculty of Theology	Intellectual history review	2,022	cc-by	11,298	Intellectual History Review ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rihr20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rihr20 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Christa Lundberg cl708@cam.ac.uk University of Cambridge Christa Lundberg To cite this article: Christa Lundberg (2022): Humanists and scholastics in early sixteenth- century Paris: new sources from the Faculty of Theology, Intellectual History Review, DOI: 10.1080/17496977.2022.2152996 To link to this article: https://doi.org/10.1080/17496977.2022.2152996 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 20 Dec 2022. Submit your article to this journal Article views: 351 View related articles View Crossmark data INTELLECTUAL HISTORY REVIEW https://doi.org/10.1080/17496977.2022.2152996 Humanists and scholastics in early sixteenth-century Paris: new sources from the Faculty of Theology Christa Lundberg Christa Lundberg St Catharine’s College, University of Cambridge, Cambridge, UK ABSTRACT Historians often compare the relationship between humanists and scholastics in the early sixteenth century to a battle. In such accounts, the Parisian Faculty of Theology plays the role of a major combatant keeping humanists away from religious studies. This article paints a diﬀerent and more harmonious picture of humanists and scholastics in the decade before the Reformation. It draws on hitherto little explored evidence from manuscripts authored by oﬃcial orators at the University of Paris: their speeches to graduating students at the Faculty of Theology in 1510 and 1512. It will be argued that the speakers celebrated both humanist and scholastic competences and the speeches themselves demonstrate that eloquence had a role to play within the institution. In this way, the article adds nuance to our understanding of how the Faculty of Theology viewed humanists and introduces important new sources to the history of universities. KEYWORDS Humanism; University of Paris; academic oratory; Faculty of Theology ABSTRACT In February 1510, twenty-ﬁve theology students gathered for one of the ﬁnal rituals before the doctoral disputations. Over four days, they met in theological colleges to receive the oﬃcial “call to the licence.” In charge of the ceremony was Olivier de Lyon, an orator appointed by the university chancellor. He addressed each student in turn, praising his learning, diligence and character in front of a large audience. De Lyon ended by formally inviting the candidate to attend another ceremony at the chan- cellor’s residence, where he would receive the licentiate. It was a celebration of the accomplishments and qualities that made the candidates worthy future members of the Faculty of Theology. The surviving graduation orations from 1510, 1512 and 1514 are previously untapped resources for considering the intellectual culture of the Faculty of Theology.1 The speeches illuminate the faculty’s perspective on theological competence – what capacities bestowed special honour on individual candidates for the doctorate – and ideas about the history and social role of theology were celebrated. Composed during the pivotal years when the Reuchlin aﬀair was deepening the controversy between graduate theolo- gians and secular scholars, these orations provide a rare insight into the changing 2 C. LUNDBERG faculty.2 Furthermore, they have the potential to counteract a source of bias against scholastics – namely, the eﬃcacy of humanist rhetoric – as a distinctly humanist style of epideictic was used to praise the candidates from the Faculty of Theology. Beyond eroding this particular source of bias, I shall argue that the speeches constitute a close parallel with a phenomenon observed by Nancy Siraisi in early modern faculties of medi- cine: humanist rhetoric contributed to reshaping the self-image of academic disciplines by celebrating their knowledge and history in new ways.3 Olivier de Lyon and Louis de Lasseré were the appointed orators in 1510 and 1512, respectively. The two men shared certain characteristics that made them suitable for the task. First, they were both active at the Collège de Navarre, which was one of the largest theological colleges. ABSTRACT In the previous century, the college had become an important humanist centre, with authors such as Jean de Montreuil, Nicolas de Clamanges and Jean Gerson aﬃliated.4 Navarre oﬀered a programme of preparatory studies in grammar and rhetoric and even provided bursaries to support students in these subjects.5 De Lyon was submagister in grammar for several years, including in 1510, when he served as orator. He additionally studied theology, achieving the bachelor’s degree before 1512 and the doc- torate in 1518.6 Lasseré had become a bachelor in theology before delivering the gradu- ation speeches in 1512. However, he did not take any higher degrees but instead devoted himself to the governance of the Collège de Navarre, where he was provisor from 1508 until 1546. De Lyon and Lasseré were both deeply entrenched in the theological commu- nity and involved in the Navarrist approach to studia humanitatis. I. The ceremonial “Call to the licence” Before moving to the graduation orations, it is worth setting the scene in more detail, especially as concerns the function and audience of the signeta ceremony. As already mentioned, the licentiate ceremony was one of the very last steps of the long path towards a doctoral degree in theology at the University of Paris. The Faculties of Law and Medicine both had equivalent ceremonies.7 In the case of theological candidates, the students had already completed their master’s in arts, their bachelor’s degree in theol- ogy and various examining disputations. The lower age limit for this degree was thirty- ﬁve years, which reveals just how long the doctoral training was. Having passed many hurdles, it was time for the candidate to receive the licentia, the papal permission to teach theology, which was bestowed by the chancellor of the university.8 In the days before the licence ceremony, the chancellor sent an orator to invite the candidates. One faculty member summarised the procedure as follows in his Compen- dium universitatis parisiensis (1517): “on the day preceding the ceremony of the License the Paranymphus delegated by the Chancellor invites in a polished speech the attendance of the candidates individually.”9 The designation of the orator as a “para- nymph” (groomsman) reﬂects the metaphorical understanding of the licence as the can- didate’s wedding to his discipline. The paranymph played the role of a ceremonial master, delivering a large number of speeches. As we shall see in the next section of this article, these speeches were no stock celebrations but strongly personalised remarks on the students’ characters and achievements, commenting on individual per- formance in disputations, academic specialisms and piety. After being praised in this way, the student in turn thanked teachers and the faculty. INTELLECTUAL HISTORY REVIEW 3 The ceremony was divided over multiple days at diﬀerent locations within Paris. The records from 1510 and 1512 suggest that the celebration migrated between four colleges: the two dominant theological colleges, Navarre and Sorbonne, and two monastic col- leges, the Dominican Couvent de Jacobins and the Franciscan Couvent des Cordeliers. That this was a standard arrangement is supported by later evidence. I. The ceremonial “Call to the licence” On 18 January 1570, twenty-seven students from the Faculty of Theology came to Parlement to invite members to attend the paranymph’s orations and outlined the schedule: Thursday at the Jacobins, Friday at the Cordeliers, at the Sorbonne on Saturday, and Navarre on Sunday.10 The invitation from 1570 raises the question of who was present at the ceremonies in the early 1510s. Although Goulet’s account from 1517 does not answer this question, he claimed that other graduation ceremonies were well attended by the general public. When the university chancellor ranked and licensed the theological candidates, accord- ing to Goulet this event attracted “nearly the whole population of Paris, not to mention the University, and a great multitude from elsewhere.”11 Similarly, the ceremony where the “Doctorate and the round magisterial biretta” were awarded attracted not only the members of the faculty but also a variety of dignitaries, including bishops, sometimes the king, cardinals, royal counsellors, and magnates and nobles invited by the doctoral candidates.12 These comments suggest that the graduation ceremonies was not a purely internal aﬀair. Throughout the sixteenth century, controversies regularly arose from inappropriate orations or unﬁtting replies from students. As James Farge discovered, one of the stu- dents graduating in 1512 was criticised by the faculty for failing to thank his teachers properly in his reply to Lasseré’s oration.13 In 1514 the doctoral candidate Jerome de Hangest was accused of having satirised the faculty member Nicolas Le Clerc.14 The same year Nicolas Cappelly was accused of insulting his regent master Jean Girard in his response to the paranymph.15 The scandals caused by inappropriate behaviour by the paranymph or students highlight the social signiﬁcance of the ritual. The praise they received, as well as their behaviour, needed to reﬂect that they were deserving of becoming full members of an important scholarly community. II. Humanist epideictic at the Faculty of Theology De Lyon and Lasseré were both involved in organising the grammatical education at Navarre, which was currently in an expansive phase.16 Lasseré had taught humaniores litterae before becoming provisor of the College in 1508.17 De Lyon taught grammar for many years and assisted the principal of the grammarians, Jean Bolu, even after embarking on his own studies in theology.18 De Lyon was particularly engaged in the project of eﬀecting a humanist turn in the curriculum, as we see in an undated letter from Guillaume Budé addressed to him. Budé wrote that he was ﬁlled with joy upon hearing about educational reforms in the grammar school at Navarre and, in particular, De Lyon’s eﬀort to teach eloquence. Budé wrote with encouragement, advising De Lyon to primarily teach ancient authors and grammarians.19 The humanist revival at Navarre provides, I shall argue, a crucial context for the interpretation of De Lyon and Lasseré’s orations. Farkas Gábor Kiss makes two relevant observations concerning the rhetoric of the paranymph orations from 1514. The ﬁrst 4 C. LUNDBERG C. LUNDBERG observation concerns the style of the speeches, which, as Kiss points out, is closely aligned with the epideictic ecclesiastical oratory studied by John O’Malley. The second point is about particular inﬂuences: Kiss found echoes of Erasmus’s Adages and the Praise of Folly in the graduation speeches, where the orator used irony and paradoxical praise.20 Drawing on the orations from 1510 and 1512, I shall next expand and contextualise these observations. Like the paranymph orations from 1514, the earlier ones ﬁt well into the model of humanist epideictic oratory described by O’Malley. Unlike in scholastic “thematic sermons,” there was no rigid division of the topic, or argumentative scheme. Instead, they relied on classical rhetorical models for structure and focalised a single point of praise, supported by many loci.21 The similarities to epideictic sermons are most striking in the speeches in praise of theology – prime examples of the genre laus disciplinae – which the paranymph delivered before turning to individual candidates. Nine such ora- tions survive from 1510–1514: four by De Lyon, three by Lasseré and two by the unknown orator of 1514. The variety of material in these speeches shows that there were no set topics: the paranymph chose which aspects of theology to praise and how. Some orations lack obvious connection to the context of graduation. II. Humanist epideictic at the Faculty of Theology For example, there are speeches on the positive impact of theology on society (1510), the soul’s immor- tality (1512), and analogies between diﬀerent kinds of theology and ﬂat, concave and convex mirrors (1514).22 However, most of the speeches were directly concerned with theology as a form of erudition and an academic discipline. gy After speaking in praise of theology, the paranymph called each candidate to the licence in a personalised speech.23 The orator praised the students’ hard work, learning, virtue and a myriad of other merits. The orations vary in length, but the general range is between 600 and 900 words. These speeches followed, roughly, the loci recommended by classical rhetoric for such speeches. They might well have used guides such as Aphtho- nius’s Progymnasmata.24 This manual suggested that encomia should discuss the person’s parental and geographical origins, upbringing, excellences of mind, body and fortune, and provide favourable comparisons. This model, as we shall see later, resulted in individualised speeches that not only provide biographical information on the gradu- ating students but also reveal the diversity of their characters. g y One common feature of the general speeches in praise of theology and of the orations to particular graduates is the frequent references to classical antiquity. De Lyon regularly employed one of the main ﬁgures of humanist sacred epideictic that O’Malley calls “quanto magis.” In short, the orator speaks about ancient culture before turning to con- sider how much better the Christian equivalent is.25 For example, De Lyon showed how much glory and honour was associated with learning in antiquity to stress how much more theological study ought to be honoured.26 In the same spirit, De Lyon’s oration to the candidate Nicolas Helm stated that he was “far happier than those ancients – Ana- xagoras, Byante, Democritus – who surrendered their riches and yet could never follow the true image of truth, which you [Nicolas] found in the garden of theology.”27 Most orations to candidates made comparisons between the candidate’s qualities and ancient characters in this way. II. Humanist epideictic at the Faculty of Theology To mention only two among very numerous examples, Lasseré claimed that Pierre Crockaert worked as hard at his studies as Pliny the Younger and in De Lyon’s speech to Jacobus Pasqueti, the candidate was compared to Caesar’s friend Labienus.28 Lasseré also mentioned Old Testament ﬁgures in this 5 INTELLECTUAL HISTORY REVIEW context – for example comparing David Cranston with the David who defeated Goliath.29 De Lyon, however, remained completely within the realm of classical antiquity. q y The many classical anecdotes not only inform us about De Lyon’s and Lasseré’s huma- nist reading preferences but also indicate what strategies they might have used in com- posing the speeches. The orators probably used material from reference books such as Valerius Maximus’s Facta et dicta memorabilia. We also have reason to believe that De Lyon and Lasseré kept their own commonplace books.30 One of the most successful commonplace book authors of the early sixteenth century, Johannes Ravisius Textor (c.1493–1522), was a colleague of theirs at Navarre. Textor, who specialised in rhetoric, regarded De Lyon as a role model and had perhaps studied with the older humanist.31 Textor published several encyclopaedic tools with excerpts from ancient literature, including Epithetorum opus (1518) and Oﬃcina (1520). The latter work contained lists of ancients (real and ﬁctional) sharing a speciﬁc virtue, vice, childhood experience, type of name, profession, cause of death etc.32 This is precisely the type of material De Lyon and Lasseré were using in their speeches. The Navarre grammarians’ eﬀort to teach literary elegance and engage with ancient authors, which Budé had praised in his letter to De Lyon, shone through in the speeches that De Lyon and Lasseré presented to the Faculty of Theology. III. Vitae scholasticae In their written form, the paranymph speeches constitute collective biographies of the graduating classes of 1510 and 1512. James Farge’s prosopography of the members of the Faculty of Theology in 1500–1536 illuminated the graduates’ geographic and social origins, religious aﬃliations, educational background and their activity in faculty delib- erations, teaching and publishing.33 The graduation orations present important additional insight into the mentality of students graduating in these same years and allow us to address questions relating to scholastic education and culture. What virtues and skills were highly valued in this community? What intellectual specialisms were represented? The brief biographies included in the graduation orations vary in the amount of detail but generally cover most of the loci recommended by rhetorical handbooks for speeches praising a person. As an example of the typical coverage, we can take De Lyon’s speech dedicated to Nicolas Ensche. First, De Lyon mentioned his place of birth near Trier, which De Lyon located at 27 degrees from the Pillars of Hercules and 99 degrees from the equator.34 According to De Lyon, Ensche was born to destitute parents. He neverthe- less managed to reach the Collège de Reims, where he studied philosophy before even- tually joining the Collège de Montaigu.35 We know from other sources that Ensche at the time of his graduation was a close collaborator of the theologian Noël Beda at Mon- taigu, where Jan Standonck in the previous decade had instituted a community for poor scholars.36 This circumstance helps explain the focus on poverty in De Lyon’s oration – a phenomenon encountered in orations to other students associated with Montaigu, including Gaspard Andree and Michael Guytard.37 At Montaigu, De Lyon tells us, Ensche constantly lectured on philosophy and theology. He commented on both recent and older texts: Martin Le Maître’s treatises De fortitudine and De temperantia 6 C. LUNDBERG 6 C. LUNDBERG C. LUNDBERG 6 from the ﬁfteenth century as well as earlier scholastic authors such as Guillaume d’Aux- erre and Robert Holcot.38 Lastly, De Lyon reported on the topics selected by Ensche for his recent disputations: the passion and poverty of Christ.39 Associated with Montaigu and lecturing on scholastic theology, Ensche is in many ways a traditional candidate. III. Vitae scholasticae In his graduating cohort, we also encounter men like Diogo de Gouveia – diplomat, later principal of the Collège de Sainte-Barbe and an early supporter of Ignatius of Loyola.40 According to De Lyon’s speech to Gouveia, he had ﬁrst been trained as an astrologer and served the Portuguese king in this capacity before being sent to Paris for further studies. After an eventful sea voyage, where Gouveia was nearly taken captive by pirates, he arrived in Paris. Like Ensche, he studied philosophy at Collège de Reims and, after another stint in Portugal, he returned to study theology.41 According to De Lyon, Gouveia performed well in the ﬁnal stage dis- putations while all the same remaining devoted to literature and good conduct and con- tinuing to develop his knowledge of astrology. Through these biographical narratives, the orators introduced and commended can- didates to the university chancellor. The speeches generally highlighted the candidates’ piety, virtue and industry. If we are to believe De Lyon and Lasseré, theology students worked day and night, ﬁlling any free time with extra reading, prayer and writing. One candidate who particularly ﬁts this bill was Noel Godefroy, whom Lasseré charac- terised as “more solitary than a Carthusian.” Lasseré further described the toll that hard work had taken on Godefroy’s body – stating that his eyelids were now drooping, his eyes retreating, his ﬂesh contracted and pale like boxwood, and the dignity of his brow was, apparently, destroyed.42 This description of Godefroy’s appearance is an unusually ruthless example of the orators’ habit of remarking on the physical appearance of the can- didate standing before them – in one case even commenting on the candidate’s expected embarrassment.43 The life stories of candidates and the paranymphs’ reports from their disputations illustrate one further aspect of their oratory style – namely, they based their praise of the candidates on argument and testimony. De Lyon and Lasseré clearly attended most disputations. III. Vitae scholasticae Both also reported on various candidates they had heard preach.44 In some cases, they referenced personal conversations with the candidate.45 Additionally, they reported the opinions of others or described how an audience had reacted to the candidate’s preaching or teaching.46 In a few cases, the testimony came from the candi- date’s students.47 Publications, such as philosophical textbooks, were also invoked as evi- dence of the candidate’s skill.48 If a candidate had received a scholarship or honour from an ecclesiastical or royal benefactor, this was reported. Other stories must have ultimately originated with the candidates themselves, such as the not infrequent accounts of child- hood poverty, the loss of parents and illness. The detailed knowledge concerning each candidate conveyed in these speeches is witness to the strong social and intellectual con- nections among advanced students of theology. A decade of studying the same texts, debating one another, and gathering for religious and academic ceremonies created a strong community. The orations demonstrate the diversity of academic specialties in the faculty. We learn that one candidate was an avid reader of Thomas Aquinas,49 that another lectured on the diﬃcult writings of the “subtle doctor” Duns Scotus,50 while yet another was an expert on the work of Bonaventure.51 Martial Mazurier, whose preaching repeatedly gave rise to 7 INTELLECTUAL HISTORY REVIEW controversy and accusations of heresy in the coming decades, was introduced as an avid student of Augustine, Jerome and Gregory.52 Whilst information concerning intellectual preferences is included in only a minority of cases, all orations comment on an essential aspect of theological education: the candi- date’s performance in the late-stage disputations before the licence.53 Disputation was central to scholastic universities for both pedagogical and intellectual reasons. Student examinations at all levels – from the bachelor’s degree in arts up to the doctorate in theol- ogy – were conducted in this way. Moreover, this highly structured and essentially col- lective mode of argumentation was also considered the superior method for ﬁnding the correct answer to a question.54 As Olga Weijers points out, there are still many gaps in our understanding of how or to what extent disputation techniques changed in early sixteenth-century universities. III. Vitae scholasticae LUNDBERG 8 C. LUNDBERG C. LUNDBERG 8 If you heard a feeble response, you showed your teeth, fumed, bit your lips, tore your beard. If not, you smoothed your brow and with your lips ﬁxated, your brow unmoving, your gaze fastened, and soles unmoving, you praised the response. If you heard a feeble response, you showed your teeth, fumed, bit your lips, tore your beard. If not, you smoothed your brow and with your lips ﬁxated, your brow unmoving, your gaze fastened, and soles unmoving, you praised the response. According to Lasseré, Cranston crushed any weak responder like David vanquished Goliath.62 This was, however, not the only way that disputations were portrayed in the para- nymph orations. Commenting on Burrey’s disputation, De Lyon said that “those who heard you seemed to hear a new Dionysius the Cistercian and besides these very rich teachings, you also had a certain sophistication (urbanitas) with many jokes and great charm.”63 De Lyon’s report about Amery’s performance was similarly colourful: No one missed how you untangled the senses and hidden interpretations of Revelations like a divine interpreter of marvellous meanings. Those present even saw John himself revived, or William of Paris, returning from the interior of the earth to the heavens.64 In these two cases, De Lyon did not represent the disputation as a combative exchange, instead praising the candidates’ solid knowledge and eloquence. These appear, in prin- ciple, like discussions of which Erasmus would approve. In these two cases, De Lyon did not represent the disputation as a combative exchange, instead praising the candidates’ solid knowledge and eloquence. These appear, in prin- ciple, like discussions of which Erasmus would approve. III. Vitae scholasticae Humanists raised various concerns about the disputation as a mode of truth-seeking, although perhaps not as unanimously as they criticised scholastic logic.55 For example, both Juan Luis Vives and Guillaume Budé thought that disputation was an important exercise albeit in need of modiﬁcation – in particular as concerned what evidence was allowed.56 Other points of criticism regarded the specialised and technical vocabulary used in scholastic disputation as well as the inherent orientation towards conﬂict. This perspective is summed up well by Erasmus, when he expressed his hope that “sober and sane discussion” would replace “sophistical and subtle disputations” in the theologi- cal faculties.57 It remains unclear to what extent such criticism changed the practice of disputation. The problem is, in part, the paucity of sources. For the Faculty of Theology in Paris, only a few published disputations survive. These include Jacques Almain’s resumptiva, argued in connection with the vesperia of his colleague Ludwig Ber in 1512.58 A third student from the same cohort, Marc de Grandval, published a version of his own vesperia the fol- lowing year.59 Almain’s and Grandval’s disputations were published because they dealt with a highly controversial and topical issue: the pro-papal arguments of Thomas Cajetan. The published disputations detail the arguments over ecclesiastical authority put forward by Almain and Grandval. However, they say little about the nature of the discussion – whether it was “sane” or “sophistical,” to use Erasmus’s terms. The para- nymph orations, by contrast, are a rich source on the subject. In the ﬁrst place, the paranymph orations inform us about some of the topics treated in disputations. I have already mentioned Ensche’s disputations on the passion and poverty of Christ. We also learn that the Cistercian monk Jean de Burrey during his ten- tativa discussed human perfection in relation to intellect, will, synderesis, charity and merit.60 Guillaume Amery’s ordinaria dealt with Revelation and the coming of the Anti- christ.61 These examples highlight how topics relating to New Testament texts and moral theology were selected alongside the themes of ecclesiastical authority treated by Almain and Grandval. Almost all the orations praise the candidates’ sharp arguments and subtle responses – commonplaces that tell us little about what actually took place during the disputations. Some, however, describe revealing details. The speech to David Cranston depicts the aggressive vibe disliked by many humanists. Lasseré described Cranston’s strong physical reaction to respondents during disputations: 8 C. IV. Scholastics and humanists in the student body The paranymph orations from 1510 to 1512 demonstrate the coexistence of scholastic approaches to theology with other traditions that were more amenable to humanists. To further explore the relationship between these diﬀerent groups, this ﬁnal section focuses on the orations dedicated to students of clear scholastic or humanistic leanings. The former group is best exempliﬁed by students from the circle of John Mair (c.1467– 1550). Mair was one of the most active teachers of nominalist philosophy and theology at the University of Paris in this period. He had studied theology with Jan Standonck and remained associated with Montaigu after receiving his licence in theology in 1506.65 Six years later, three of Mair’s students received the licence: David Cranston, Jacques Almain and Pierre Crockaert. Their success at the Faculty of Theology is indicated by the ranking that teachers at the faculty made of the candidates in the licentiate class. The oﬃcial ranking of candidates in 1512 placed all three highly: Almain was ranked second in the class (after Ludwig Ber), Cranston ﬁfth and Crockaert sixth. Association with one of the leading scholastic theologians was clearly correlated with success in the Faculty of Theology. The three candidates had all published works in the scholastic tradition of philosophy. Cranston had published on logic and physics; Almain on logic, physics and ethics; and Crockaert had written works on logic and Thomist philosophy. In this activity too they followed Mair, who published many books throughout his career. His early publi- cations focused mainly on logic, but he later wrote commentaries on the Sentences (from 1509 onwards), a Gospel commentary (1518) and a work on British history (1521). It seems likely that the three candidates presented to the university chancellor in 1512 would have followed in Mair’s footsteps as proliﬁc writers in the scholastic tra- dition, had not all three died in the years immediately following their graduation.66 We have already seen that Lasseré’s speech to Cranston thematised his combative per- formance in disputations. This portrait of Cranston resonates with how Mair himself INTELLECTUAL HISTORY REVIEW 9 depicted his student as a staunch defender of traditional scholastic method. In 1510, Mair made Cranston one of the interlocutors of a short dialogue published as a preface to his own commentary on the ﬁrst book of the Sentences. In the dialogue, Cranston undertook discussion with a humanist critic of Mair’s method. IV. Scholastics and humanists in the student body Cranston defended the use of Aris- totle and philosophical concepts in theology. Furthermore, he argued that for solving complex questions it is necessary to pay sustained attention to arguments pro and contra according to the scholastic method.67 The willingness to dialogue with humanists is telling of Mair’s openminded attitude. It has been pointed out that Mair attended some classes in Greek and that he in part sympathised with the views of humanist educational reformers. In 1528 Mair himself suggested that theologians had perhaps spent too much time on philosophy and that it was time to engage more closely with Scripture.68 The early dialogue ends openly, without a clear sign that Cranston successfully convinced his opponent. Yet the methodology employed in Mair’s Sentences commentary made his own preference abundantly clear. The awareness of Mair’s circle as a bastion of scholastic traditionalism is evident in Lasseré’s orations to Almain and Crockaert. Lasseré’s speech to Almain focused almost exclusively on his intellectual achievements. The orator praised Almain’s capacious memory, his successful teaching at the Collège de Coqueret and the dialectical works written on sleepless nights.69 In describing Almain’s work, Lasseré emphasised his ability to resolve complex problems and explain the most obscure and diﬃcult matters lucidly: “nothing could be said more clearly or easily than in your Sorbonic disputa- tions.”70 In his speech to Crockaert, Lasseré thematised Crockaert’s turn to Thomist phil- osophy following his entry into the Dominican order, praising his ability to explain both vias – the nominalist and the realist – and comparing him to Thomas as well as Durand de Saint-Pourçain.71 Lasseré furthermore remarked that Crockaert did not write in frivo- lous genres like poetry, history or satire but “in subtle windings like Aristotle, and salu- tary warnings like Paul.”72 Lasseré’s speeches to theologians of the scholastic camp incorporated praise sympathetic to their point of view. The same is true of Lasseré’s speeches to candidates with clear humanistic allegiances. Among the candidates celebrated in 1512 was Valerand de La Varanne, an accomplished poet. Lasseré especially praised La Varanne’s patriotic Carmen de expugnatione genuensi from 1507.73 His speech, however, opened with a more general defence of the liberal arts, arguing that “the theologian should know many things besides theology.”74 In the speech, Lasseré presented various traditional arguments for why grammatical and rhetorical knowledge was relevant for the theologian. First, the seculares scientiae add ornamenta- tion to divine letters. IV. Scholastics and humanists in the student body Second, liberal disciplines had been essential to authors like Lactan- tius and Augustine. Their erudition had allowed Lactantius to “tear down” the superstitions of pagans, and Augustine to artfully erect the “city of God.” Therefore, we should not listen to people who “having ﬁnished their study of theology strive to dis- suade people from the knowledge of liberal arts.”75 Lasseré ended his speech to La Varanne with an exhortation for the multiscius to rise and rejoice – echoing the ideal of encyclopaedic knowledge embraced by many French Renaissance authors.76 y g y y Several students and orators had connections to the philosopher and humanist edu- cational reformer Jacques Lefèvre d’Étaples (c.1460–1536).77 La Varanne probably knew Lefèvre through common friends at the Collège de Boncourt or from the Picard nation.78 In 1508, he published a poem in Lefèvre’s honour. The poem highlighted the 0 C. LUNDBERG C. LUNDBERG 10 religious implications of Lefèvre’s approach to philosophy, which La Varanne character- ised as a kind of natural theology. By investigating the creation – in particular “its hidden corners” (abdita) – Lefèvre was learning about God.79 La Varanne ended by encouraging Lefèvre’s contributions to learning in Paris.80 De Lyon similarly praised Lefèvre’s approach to philosophy in an oration, highlighting again that theology students with humanistic interests were not averse to these ideas. This segment is found in De Lyon’s speech to Philippe Prevost. Prevost had originally studied with a diﬀerent teacher but after gaining the master’s degree, he taught philosophy along- side Lefèvre at the Collège de Cardinal Lemoine, where he was also a bursar in theology.81 In 1503 Prevost had received a friendly dedication from Lefèvre’s close collaborator Josse Clichtove, designating him a “companion in the study of philosophy” (in philosophiae studio commilitonus).82 Reﬂecting on Prevost’s path, De Lyon told this story as a passage from sophism to true philosophy: … at the beginning you had been taught and shaped in those schools where you encountered sophistical fallacies and fallacious sophisms, where you tasted Aristotle, as they say, “with the edge of your lips.” Having true philosophy ﬁxed to your heart with great spikes, with regret you were led towards the most learned Lefèvre who – if I may use the words of Plau- tinus – set a ruler to Aristotle’s books, and brought back home the peripatetic Aristotle, which had been obfuscated by certain labyrinths and puzzles … . 1. Olivier de Lyon’s orations from 1510 survive in two manuscripts in the Biblioteca Apostolic Vaticana [BAV]: MSS Reg. lat. 701 and 1373. Louis de Lasseré’s orations from 1512 survive in three copies: British Library [BL] MS Harley 2536 and Bibliothèque nationale de France [BNF] MSS Latin 7812 and 7813. Some speeches by the unidentiﬁed orator of 1514 are found together with various sermons and letters in Metropolitan chapter MS 832 in Prague, see Kiss, “‘O Pragensis Achademia!’”. I am grateful to Dr. Kiss for sharing images of this manuscript with me. Besides these six manuscripts I am not aware of any further sur- viving paranymph orations from the ﬁrst half of the sixteenth century. IV. Scholastics and humanists in the student body In one of his orations on theology, De Lyon addressed the question of how theology related to philosophy: … the theology that investigates the cause of causes is the highest form of philosophy; the theology that deﬁnes the obligations of virtues in their circumstances is the highest form of ethics; the theology that teaches indestructible truth is the highest form of logic … .89 In sum, De Lyon’s orations show that Lefèvre’s vision of theologising philosophy gained support beyond his closest students and members of his college, even reaching students at the Faculty of Theology. In sum, De Lyon’s orations show that Lefèvre’s vision of theologising philosophy gained support beyond his closest students and members of his college, even reaching students at the Faculty of Theology. I shall conclude with two points relating to the scholastic and humanist students at the Faculty of Theology. The ﬁrst relates to the ranking of students. I have already mentioned the high rankings achieved by the students from John Mair’s circle. Cranston, Almain and Crockaert were among the top students at the faculty. By contrast, Lamy ranked 19/29 and Prevost 26/29.90 While all these students were praised for their philosophical skill by the paranymphs, it is clear that the masters of the faculty preferred Mair’s students. Second, the graduation speeches from the Faculty of Theology in 1510–1512 open up a new perspective on the relationship between humanists and theologians other than the confrontational narrative embraced by most recent studies. For example, Ann Moss argues that humanists and scholastics in Paris and elsewhere belonged to separate linguistic spheres, between which little, if any, communication or compromise was possible.91 James Farge argues that the members of the Faculty of Theology shared a conservative mentality that put them completely at odds with humanist innovators and reformers.92 These narratives ﬁt well with the testimony of some, for whom a humanistic education appeared incompatible with the culture of the faculty. However, many students saw no obvious contradiction between the academic study of theology and humanist learning. In the decade before the Reformation, studia humanitatis still played an important and constructive role within the Faculty of Theology. IV. Scholastics and humanists in the student body You applied yourself with so much eﬀort that you emerged most skilled among those who stepped out of Lefèvre’s shadow.83 De Lyon’s speech to Provost thus presented sympathetically the Fabrist narrative con- cerning humanist philosophy. He discussed the philological project of cleansing the text, and the approach that was more generally concerned with avoiding the tricky sophistical problems associated with certain branches of scholasticism. Moreover, he said, this approach to Aristotle uncovered the “most concealed places of philosophy” – a line of praise borrowed from Francesco Pucci’s letter regarding Angelo Poliziano’s encyclopaedic Miscellanies.84 Two further passages from De Lyon’s orations contain close parallels to Lefèvre’s ideas concerning the relationship between philosophy and theology. One is found in his oration to the otherwise little-known theologian Nicolas Lamy. Lamy had taught philos- ophy for many years at the Collège de Calvy and particularly studied natural philosophy and mathematics, including Archimedes’s spheres, Democritus’s atoms and Pythagoras’s numbers.85 Like Charles de Bovelles and other members of Lefèvre’s circle, Lamy paired his philosophical study with contemplative practices.86 Playing on the Platonist notion of the soul’s celestial origin, De Lyon said: Just as the endless God the Father gave you a soul from the eternal ﬁres, which we call stars and constellations, so it seems that you, always attending to the heavens and the celestial fatherland, are frequently raptured into heavens through contemplation.87 According to De Lyon, Lamy’s capacity for rapture and ecstasy made him a welcome visitor in reformed monasteries, where he would preach about God and the “council” of the heavens. The same combination of devotion and erudition explained, according to De Lyon, why Lamy had been made prior of the Sorbonne.88 De Lyon’s description of Lamy’s contemplation echoes Fabrist views on the relation- ship between philosophical knowledge and religious insight: studying and looking 1 11 INTELLECTUAL HISTORY REVIEW 11 towards the “blessed region” might invite divine illumination. We unfortunately have no surviving writings by Lamy to corroborate that he shared their outlook. De Lyon himself, however, was clearly sympathetic to Lefèvre’s views. f 4. Ouy, “Le Collège de Navarre”. 3. See her discussion of the orations of Jean Le Vieil (1560) and those of Gabriel Naudé from the early 1600s in Siraisi, “Oratory and Rhetoric in Renaissance Medicine”; Siraisi, History, Medicine, and the Traditions of Renaissance Learning, 127–32. g p y p y 2. Rummel, The Case Against Johann Reuchlin; Farge, “Noël Beda and the Defense of the Tra- dition”, 148. f g 4. Ouy, “Le Collège de Navarre”. Notes 1. Olivier de Lyon’s orations from 1510 survive in two manuscripts in the Biblioteca Apostolic Vaticana [BAV]: MSS Reg. lat. 701 and 1373. Louis de Lasseré’s orations from 1512 survive in three copies: British Library [BL] MS Harley 2536 and Bibliothèque nationale de France [BNF] MSS Latin 7812 and 7813. Some speeches by the unidentiﬁed orator of 1514 are found together with various sermons and letters in Metropolitan chapter MS 832 in Prague, see Kiss, “‘O Pragensis Achademia!’”. I am grateful to Dr. Kiss for sharing images of this manuscript with me. Besides these six manuscripts I am not aware of any further sur- viving paranymph orations from the ﬁrst half of the sixteenth century. g p y p y 2. Rummel, The Case Against Johann Reuchlin; Farge, “Noël Beda and the Defense of the Tra- dition”, 148. 3. See her discussion of the orations of Jean Le Vieil (1560) and those of Gabriel Naudé from the early 1600s in Siraisi, “Oratory and Rhetoric in Renaissance Medicine”; Siraisi, History, Medicine, and the Traditions of Renaissance Learning, 127–32. f 4. Ouy, “Le Collège de Navarre”. 12 C. LUNDBERG 5. The popularity of this program is suggested by the grammarians’ expansion into a new, larger building in 1514. On this expansion and an estimate for student numbers during the sixteenth century, see Compère, “Navarre”, 280–82. 5. The popularity of this program is suggested by the grammarians’ expansion into a new, larger building in 1514. On this expansion and an estimate for student numbers during the sixteenth century, see Compère, “Navarre”, 280–82. y p 6. Farge, Students and Teachers at the University of Paris, 36; Farge, Biographical Register, 290–91. 7 S l t l f th F lt f M di i di d i Si i i “O t d 6. Farge, Students and Teachers at the University of Paris, 36; Farge, Biographical Register, 290–91. . Some later examples from the Faculty of Medicine are discussed in Siraisi, “Oratory an Rhetoric in Renaissance Medicine”. 7. Some later examples from the Faculty of Medicine are discussed in Siraisi, “Oratory and Rhetoric in Renaissance Medicine”. 8 F O th d d R f 24 26 8. Farge, Orthodoxy and Reform, 24–26. 9. Translation cited from Goulet, Compendium on the University of Paris, 60. On Goulet, see Farge, Biographical Register, 201–2. 9. Translation cited from Goulet, Compendium on the University of Paris, 60. Notes On Goulet, see Farge, Biographical Register, 201–2. g g g 10. Cited in Du Boulay, Historia Universitatis Parisiensis, 6:709. See also Farge, Farge, Ortho- doxy and Reform, 25, n. 81. 10. Cited in Du Boulay, Historia Universitatis Parisiensis, 6:709. See also Farge, Farge, Ortho- doxy and Reform, 25, n. 81. y f 11. Translation cited from Goulet, Compendium on the University of Paris, 59. l 12. Goulet, 60. 13. Farge, Orthodoxy and Reform. Complaints about the “ingratitudo licentiandorum” were ﬁrst raised at a Faculty meeting in February 1512. The aﬀair can be followed in Clerval, Registre des procès-verbaux, 103–5. 14. Farge, Biographical Register, 218. 15. Farge, 65. 16. There is little modern literature on the topic but we now have a good starting-point in Nathaël Istasse’s work, see Istasse, Joannes Ravisius Textor; Istasse, “Pour une contribution à l’histoire de l’enseignement du latin à la Renaissance”. 17. Launoy, Regii Navarrae gymnasii Parisiensis historia, 1:676. y g gy 18. Lyon was submagister grammaticorum, as he writes in the “ex dono” in BAV Reg. lat. 1373. On Bolu, see Farge, Biographical Register, 50–51; Istasse, Joannes Ravisius Textor, 59–62. 19. Budé, Opera omnia, 1:392–93. 18. Lyon was submagister grammaticorum, as he writes in the “ex dono” in BAV Reg. lat. 1373. On Bolu, see Farge, Biographical Register, 50–51; Istasse, Joannes Ravisius Textor, 59–62. 19. Budé, Opera omnia, 1:392–93. 19. Budé, Opera omnia, 1:392–93. 20. Kiss, “‘O Pragensis Achademia!’”, 163–64. Kiss discusses the graduation speeches alongside various other sermons found in this fascinating manuscript. 20. Kiss, “‘O Pragensis Achademia!’”, 163–64. Kiss discusses the graduation speeches alongside various other sermons found in this fascinating manuscript. 21. On what distinguished humanist epideictic from its scholastic counterpart, see O’Malley, Praise and Blame in Renaissance Rome, 50–76. 21. On what distinguished humanist epideictic from its scholastic counterpart, see O’Malley, Praise and Blame in Renaissance Rome, 50–76. 22. BAV Reg. lat. 1373, 80–85; BNF Latin 7812, 31v–37; Prague, Metropolitan chapter 832, 1r–v. The latter theme betrays the inﬂuence of Cusanus, possibly via his Parisian followers in the circle of Lefèvre, see Rice, The Prefatory Epistles, 342–48. 22. BAV Reg. lat. 1373, 80–85; BNF Latin 7812, 31v–37; Prague, Metropolitan chapter 832, 1r–v. The latter theme betrays the inﬂuence of Cusanus, possibly via his Parisian followers in the circle of Lefèvre, see Rice, The Prefatory Epistles, 342–48. 23. Notes BAV Reg. lat. 1373, 67v. Farge, Biographical Register, 318–22. 52. BAV Reg. lat. 1373, 67v. Farge, Biographical Register, 318–22. 53. See Farge, Orthodoxy and Reform, 22–26. 54. A good overview of the use of disputation in medieval universities is Weijers, In Search of the Truth, 119–47. 54. A good overview of the use of disputation in medieval universities is Weijers, In Search of the Truth, 119–47. 54. A good overview of the use of disputation in medieval universities is Weijers, In Search of the Truth, 119–47. 55. Kristeller, Medieval Aspects of Renaissance Learning, 10. 55. Kristeller, Medieval Aspects of Renaissance Learning, 10. 56. See Weijers, In Search of the Truth, 189–91. See also Sayhi-Périgot, Dialectique et littérature. 56. See Weijers, In Search of the Truth, 189–91. See also Sayhi-Périgot, Dialectique et littérature. j , f , y g , q 57. Erasmus, Opus epistolarum Ep. 1111. “pro sophisticis argutationibus nunc sobrias ac sanas inter theologos disputationes” 57. Erasmus, Opus epistolarum Ep. 1111. “pro sophisticis argutationibus nunc sobrias ac sanas inter theologos disputationes” 58. A revised text was published in 1512, see Almain, Libellus de auctoritate ecclesie. On the version of Almain’s resumptiva published in 1518, see Almain, “Question at Vespers”. d d l d 58. A revised text was published in 1512, see Almain, Libellus de auctoritate ecclesie. On the version of Almain’s resumptiva published in 1518, see Almain, “Question at Vespers”. 58. A revised text was published in 1512, see Almain, Libellus de auctoritate ecclesie. On the version of Almain’s resumptiva published in 1518, see Almain, “Question at Vespers”. 9 d G d l C d i 59. de Grandval, Codex vesperiarum. 60. BAV Reg. lat. 1373, 31r. 61. BAV Reg. lat. 1373, 65r–v. 62. BNF Latin 7812, 27v. “Si futilem responsionem audires ringebas, stomachabare, labra mordebas, barbam vellebas, si minus frontem exporrigebas, ﬁxis labris, immoto supercilio, conﬁxis oculis, immotis vestigiis, responsionem laudabas, qui si respondentem per negationem assumpti erectum oﬀendebas solerti probatione collisum confractumque reddebas ut aliter David … .”. 62. BNF Latin 7812, 27v. “Si futilem responsionem audires ringebas, stomachabare, labra mordebas, barbam vellebas, si minus frontem exporrigebas, ﬁxis labris, immoto supercilio, conﬁxis oculis, immotis vestigiis, responsionem laudabas, qui si respondentem per negationem assumpti erectum oﬀendebas solerti probatione collisum confractumque reddebas ut aliter David … .”. 63. BAV Reg. lat. Notes “ubi continuis lectionibus ingenium tuum ne torpesceret exercuisti modo philosophiam modo theologiam proﬁtens, modo martinum de fortitudine; de temper- anaia modo gabrielem altissiodorensis alias olkot interpretans.” De Lyon presumably meant to refer to the scholastic theologian Guillaume of Auxerre and not “Gabriel of Auxerre”. 39 BAV Reg lat 100v 38. BAV Reg. lat. 1373, 100r. “ubi continuis lectionibus ingenium tuum ne torpesceret exercuisti modo philosophiam modo theologiam proﬁtens, modo martinum de fortitudine; de temper- anaia modo gabrielem altissiodorensis alias olkot interpretans.” De Lyon presumably meant to refer to the scholastic theologian Guillaume of Auxerre and not “Gabriel of Auxerre”. 39 BAV Reg lat 100v 38. BAV Reg. lat. 1373, 100r. “ubi continuis lectionibus ingenium tuum ne torpesceret exercuisti modo philosophiam modo theologiam proﬁtens, modo martinum de fortitudine; de temper- anaia modo gabrielem altissiodorensis alias olkot interpretans.” De Lyon presumably meant to refer to the scholastic theologian Guillaume of Auxerre and not “Gabriel of Auxerre”. 39. BAV Reg. lat. 100v. 40. Farge, Biographical Register, 202–4. 41. BAV Reg. lat. 1373, 136r–140v. 42. BNF Latin 7812, 52v: “Nam labore assiduo, comite abstinentia laxantur gene, subintrant oculi, buxo pallidior caro contrahitur, frontis deperditur dignitas, ut te universum usque adeo iuverit libros heluari et indefessis animis chartis impalescere.” 42. BNF Latin 7812, 52v: “Nam labore assiduo, comite abstinentia laxantur gene, subintrant oculi, buxo pallidior caro contrahitur, frontis deperditur dignitas, ut te universum usque adeo iuverit libros heluari et indefessis animis chartis impalescere.” 42. BNF Latin 7812, 52v: “Nam labore assiduo, comite abstinentia laxantur gene, subintrant oculi, buxo pallidior caro contrahitur, frontis deperditur dignitas, ut te universum usque adeo iuverit libros heluari et indefessis animis chartis impalescere.” 43. BNF Latin 7812, 86 r–v. On the preference for visual description and language in humanist epideictic, see O’Malley, Praise and Blame in Renaissance Rome, 63. 43. BNF Latin 7812, 86 r–v. On the preference for visual description and language in humanist epideictic, see O’Malley, Praise and Blame in Renaissance Rome, 63. 43. BNF Latin 7812, 86 r–v. On the preference for visual description and language in humanist epideictic, see O’Malley, Praise and Blame in Renaissance Rome, 63. 44. BAV Reg. lat. 1373, 102v–103v. 45. BAV Reg. lat. 1373, 50v–51r. 46. BAV Reg. lat. 1373, 17v–18r. 47. BNF Latin 7812, 82v. 48. Prague, Metropolitan chapter 832, 6v. 49. BAV Reg. lat. 85v; BNF Latin 7812, 29v–30r. 50. BNF Latin 7812, 67v. 51. BAV Reg. lat. 1373, 33v. 52. Notes With very few exceptions, the candidates who received the license also received the docto- rate and are included in Farge’s register of graduates, see Farge, Biographical Register. 24. Margolin, “La rhétorique d’Aphthonius et son inﬂuence au XVIe siècle”. 25. See O’Malley, Praise and Blame in Renaissance Rome, 57. 26. BAV Reg. lat. 1373, 107v. 27. BAV Reg. lat. 1373, 53r: “ … ad eam tibi comparandam in hoc longe beatior illis antiquis – anaxagora, byante, democryte – qui sese opibus sequestrarunt et tamen veram veritatis yma- ginem assequi nunquam potuere, qua in orto theologo abste inventa inimicas ac anxias abii- cere voluptates que animam sibi vinciunt, eterna preferre brevibus utila iocundis didicisti: proinde nichil gratum tibi est, nisi quod iuste, quod pie sit nichil auditu suave nisi quod animam teque meliorem reddit.” 28. BNF Latin 7812, 13v–14; BAV Reg. lat. 1373, 133v–134r. 28. BNF Latin 7812, 13v–14; BAV Reg. lat. 1373, 133v–134r. 29. BNF Latin 7812, 27v. 29. BNF Latin 7812, 27v. 30. Moss, Printed Commonplace-Books and the Structuring of Renaissance Thought; Blair, “The Rise of Note-Taking in Early Modern Europe”. 30. Moss, Printed Commonplace-Books and the Structuring of Renaissance Thought; Blair, “The Rise of Note-Taking in Early Modern Europe”. 31. Istasse, Joannes Ravisius Textor, 62–64. 32. Ong, “Commonplace Rhapsody: Ravisius Textor, Zwinger and Shakespeare”; Istasse, Joannes Ravisius Textor. See also Istasse’s discussion of a manuscript relating to Textor’s teaching at Navarre in 1516: Istasse, “Pour une contribution à l’histoire de l’enseignement du latin à la Renaissance”. 32. Ong, “Commonplace Rhapsody: Ravisius Textor, Zwinger and Shakespeare”; Istasse, Joannes Ravisius Textor. See also Istasse’s discussion of a manuscript relating to Textor’s teaching at Navarre in 1516: Istasse, “Pour une contribution à l’histoire de l’enseignement du latin à la Renaissance”. 33. Farge, Orthodoxy and Reform, 55–114. INTELLECTUAL HISTORY REVIEW 13 34. BAV Reg. lat. 1373, 99r. g 35. Documents from the German Nation give a more positive view of Ensche’s personal ﬁnances, cf. Farge, Biographical Register, 155. 35. Documents from the German Nation give a more positive view of Ensche’s personal ﬁnances, cf. Farge, Biographical Register, 155. 35. Documents from the German Nation give a more positive view of Ensche’s personal ﬁnances, cf. Farge, Biographical Register, 155. 36. Compère, “Montaigu”; Bakker, “The Statutes of the College de Montaigu”. 37. BNF Latin 7812, 16r–17r, 55v–56r. 38. BAV Reg. lat. 1373, 100r. Notes “Et nostram studiis auge melioribus urbem … ” g 81. Prevost contributed verse to some of Lefèvre’s early Aristotelian editions, see Rice, 80 and 103. He was the dedicatee of another philosophical treatise during his three-month stint as rector of the University, see Francus Vimacuus, Tres Hecatonomie de conceptibus. h f l 81. Prevost contributed verse to some of Lefèvre’s early Aristotelian editions, see Rice, 80 and 103. He was the dedicatee of another philosophical treatise during his three-month stint as rector of the University, see Francus Vimacuus, Tres Hecatonomie de conceptibus. 81. Prevost contributed verse to some of Lefèvre’s early Aristotelian editions, see Rice, 80 and 103. He was the dedicatee of another philosophical treatise during his three-month stint as rector of the University, see Francus Vimacuus, Tres Hecatonomie de conceptibus. 82. Rice, The Prefatory Epistles, 110–12. 82. Rice, The Prefatory Epistles, 110–12. 83. BAV Reg. lat. 1373, 89r–v: “Cum enim initio formatus institutusque fuisses in illis scholiis ubi sophisticis captionibus captiosisque sophismatibus intentus eras, ubi verum aristotelem primoribus ut aiunt labris tantum degustabas; veram philosophiam ﬁxam cordi: habens etiam trabalibus clavis penitentia ductus es et ad eruditissimum stapulensem qui aristoteli- cos libros, ut plautino utar verbo, amussitavit peripathethicumque aristotelem mean[r]dis griphisque quibusdam obtenebratum postlimino revocavit, conversus reconditissimos phi- losophie locos et sanctius illud orarium [aerarium] unde nihil communi percussum moneta, nihil triviale, nihil conculcatum eﬀertur. Sed quasi ex aphrica semper aliquid novi prodit, adiisti tanto adhibito labore: ut inter eos qui ex stapulensibus umbraculis doctissimi pro- diere tu peritissimus evaseris.” 83. BAV Reg. lat. 1373, 89r–v: “Cum enim initio formatus institutusque fuisses in illis scholiis ubi sophisticis captionibus captiosisque sophismatibus intentus eras, ubi verum aristotelem primoribus ut aiunt labris tantum degustabas; veram philosophiam ﬁxam cordi: habens etiam trabalibus clavis penitentia ductus es et ad eruditissimum stapulensem qui aristoteli- cos libros, ut plautino utar verbo, amussitavit peripathethicumque aristotelem mean[r]dis griphisque quibusdam obtenebratum postlimino revocavit, conversus reconditissimos phi- losophie locos et sanctius illud orarium [aerarium] unde nihil communi percussum moneta, nihil triviale, nihil conculcatum eﬀertur. Sed quasi ex aphrica semper aliquid novi prodit, adiisti tanto adhibito labore: ut inter eos qui ex stapulensibus umbraculis doctissimi pro- diere tu peritissimus evaseris.” 84. De Lyon’s praise of Lefèvre’s philosophy is adapted from a letter from Francesco Pucci to Angelo Poliziano published in book VI of Poliziano’s correspondence. See Poliziano, Illu- strium virorum epistolae sig. f3v–f4v. 84. Notes BNF Latin 7812, 88v–89r: “ut nichil omnino dici possit duabus sorbonicis tuis apertius atque acilius.” 71. BNF Latin 7812, 14v. 72. BNF Latin 7812, 15v: “decantas non carmina ut Empedocles, non dyalogos ut Plato, non hymnos ut Socrates, modos ut Epicharmus, ut Xenophon historias, ut Xenocrates satiras, sed argutissimos meandros ut Aristoteles, salutaria monita ut Paulus.” 72. BNF Latin 7812, 15v: “decantas non carmina ut Empedocles, non dyalogos ut Plato, non hymnos ut Socrates, modos ut Epicharmus, ut Xenophon historias, ut Xenocrates satiras, sed argutissimos meandros ut Aristoteles, salutaria monita ut Paulus.” 73. On this poem, see Provini, “La poésie héroïque”. In the printed edition from 1507, La Varanne included shorter verses composed for various people, including Gillis van Delft, Lefèvre d’Étaples, and Geoﬀrey Boussard. 74. BNF Latin 7812, 20v. 75. BNF Latin 7812, 20v–21r: “Theologum oportet preter theologiam multa nosse. […] Exurge igitur qui multiscius es et letare.” 75. BNF Latin 7812, 20v–21r: “Theologum oportet preter theologiam multa nosse. […] Exurge igitur qui multiscius es et letare.” 76. On “the encyclopaedic paradigm” among French humanists in this period, see Pédeﬂous and Tournoy, “Juan Luis Vives and His Dialogue ‘Sapiens’”, 255–57. Encyclopaedism is also thematised in the speech to Jérome de Hangest in Prague, Metropolitan chapter 832, 6r. 76. On “the encyclopaedic paradigm” among French humanists in this period, see Pédeﬂous and Tournoy, “Juan Luis Vives and His Dialogue ‘Sapiens’”, 255–57. Encyclopaedism is also thematised in the speech to Jérome de Hangest in Prague, Metropolitan chapter 832, 6r. 77. Rice, “Humanist Aristotelianism in France”. On Lefèvre’s educational reform, see Oos- terhoﬀ, Making Mathematical Culture. 77. Rice, “Humanist Aristotelianism in France”. On Lefèvre’s educational reform, see O terhoﬀ, Making Mathematical Culture. 78. Farge, Biographical Register, 243. 79. Cited from Rice, The Prefatory Epistles, 179–80. “Mens tua dum assurgit, caelos introspicis et quod / Daedala naturae dextera ﬁngit opus. / Ut propius verum subscribam, doctus es ipse / Abdita naturae, doctus es ipse Deum.” 79. Cited from Rice, The Prefatory Epistles, 179–80. “Mens tua dum assurgit, caelos introspicis et quod / Daedala naturae dextera ﬁngit opus. / Ut propius verum subscribam, doctus es ipse / Abdita naturae, doctus es ipse Deum.” 79. Cited from Rice, The Prefatory Epistles, 179–80. “Mens tua dum assurgit, caelos introspicis et quod / Daedala naturae dextera ﬁngit opus. / Ut propius verum subscribam, doctus es ipse / Abdita naturae, doctus es ipse Deum.” 80. Rice, 180. Notes 1373, 31r: “Qui te audiebant alterum dionisium cicterciensis audire videban- tur habes preter hanc fecundissimam doctrinam faceciam quandam urbanitatemque multis Iocis multa suavitate.” On the identity of Dionysius the Cistercian, see Brinzei and Schabel, “Les Cisterciens de l’université”. 63. BAV Reg. lat. 1373, 31r: “Qui te audiebant alterum dionisium cicterciensis audire videban- tur habes preter hanc fecundissimam doctrinam faceciam quandam urbanitatemque multis Iocis multa suavitate.” On the identity of Dionysius the Cistercian, see Brinzei and Schabel, “Les Cisterciens de l’université”. 64. BAV Reg. lat. 1373, 65r–v: “certamen quam brevissime ﬁniveris in maiore ordinaria quem subtiliter antichristi adventum tractaveris; nemo est qui non norit ubi sensa abditasque apocalipsis interpretationes quasi divinus interpres miriﬁcis sensibus enodabas admiranti- bus qui aderant ac si ioannem redivivum inspicerent aut guillermum parisiensem e terre vis- ceribus ad superos redeuntem.” 64. BAV Reg. lat. 1373, 65r–v: “certamen quam brevissime ﬁniveris in maiore ordinaria quem subtiliter antichristi adventum tractaveris; nemo est qui non norit ubi sensa abditasque apocalipsis interpretationes quasi divinus interpres miriﬁcis sensibus enodabas admiranti- bus qui aderant ac si ioannem redivivum inspicerent aut guillermum parisiensem e terre vis- ceribus ad superos redeuntem.” 64. BAV Reg. lat. 1373, 65r–v: “certamen quam brevissime ﬁniveris in maiore ordinaria quem subtiliter antichristi adventum tractaveris; nemo est qui non norit ubi sensa abditasque apocalipsis interpretationes quasi divinus interpres miriﬁcis sensibus enodabas admiranti- bus qui aderant ac si ioannem redivivum inspicerent aut guillermum parisiensem e terre vis- ceribus ad superos redeuntem.” 65. See literature and bibliography in Farge, Biographical Register, 304–11. Among the more recent literature on John Mair, see Broadie, The Circle of John Mair; Slotemaker and Witt, A Companion to the Theology of John Mair. 65. See literature and bibliography in Farge, Biographical Register, 304–11. Among the more recent literature on John Mair, see Broadie, The Circle of John Mair; Slotemaker and Witt, A Companion to the Theology of John Mair. 14 C. LUNDBERG 14 66. For their publications, see Farge, Biographical Register. Cranston died in 1512, Crockaert in 1514, and Almain in 1515. d “ l d h l d ” 66. For their publications, see Farge, Biographical Register. Cranston died in 1512, Crockaert in 1514, and Almain in 1515. 67. Broadie, “Dialogus de Materia Theologo Tractanda”. 67. Broadie, “Dialogus de Materia Theologo Tractanda”. 68. On Mair’s (limited) sympathy with humanists, see Farge, Biographical Register, 307 69 BNF L i 812 8 69. BNF Latin 7812, 87v. Notes De Lyon’s praise of Lefèvre’s philosophy is adapted from a letter from Francesco Pucci to Angelo Poliziano published in book VI of Poliziano’s correspondence. See Poliziano, Illu- strium virorum epistolae sig. f3v–f4v. p g 85. Lamy was already dead in June 1513 when one of his students from Calvy sought to certify his studies, see Farge, Students and Teachers at the University of Paris, 392–93. db “Th k f h l h ” 85. Lamy was already dead in June 1513 when one of his students from Calvy sought to certify his studies, see Farge, Students and Teachers at the University of Paris, 392–93. 86. Lundberg, “The Making of a Philosopher”. 87. BAV Reg. lat. 1373, 131v–132r: “Atque tanquam inﬁnibilis pater deus animum tibi ex sem- piternis ignibus que nos sydera stellasque nuncupamus indiderit ad celos celestemque patriam semper intentum contemplationibus frequenter raptus in celo versari videris.” 87. BAV Reg. lat. 1373, 131v–132r: “Atque tanquam inﬁnibilis pater deus animum tibi ex sem- piternis ignibus que nos sydera stellasque nuncupamus indiderit ad celos celestemque patriam semper intentum contemplationibus frequenter raptus in celo versari videris.” 88. BAV Reg. lat. 1373, 132r: “Quas ut licentius oportuniusque habere possis, religiones et con- ventus reformatissimos adiens cum ipsis religionis de summo deo de celesti curia sermonem facis: vel solus tecum raptus quasi in extasi rationaris. Hoc est exercitium tuum hec est animi oblectatio a studio et oratione in contemplationem rapi. et sic brevi fecisti ut religiosam 88. BAV Reg. lat. 1373, 132r: “Quas ut licentius oportuniusque habere possis, religiones et con- ventus reformatissimos adiens cum ipsis religionis de summo deo de celesti curia sermonem facis: vel solus tecum raptus quasi in extasi rationaris. Hoc est exercitium tuum hec est animi oblectatio a studio et oratione in contemplationem rapi. et sic brevi fecisti ut religiosam 15 15 INTELLECTUAL HISTORY REVIEW animi devotionem litteraturamque non vulgarem, sed eminentissimam quod viaticum est senectutis tibi comparaveris. Quibus rationibus adducti socii sorbonici anno isto te in priorem suum elegerunt.” The ms says “rationaris” but this should surely be ratiocinaris. Themes of rapture and ecstasy are not uncommon in the orations, for another example see Reg. lat. 1373, 32v–33. 89. BAV Reg. lat. 1373, 108v–109r. “theologiam, inquam, que causam causarum discutiens, summa est philosophia; que virtutum oﬃcia suis circumstantiis diﬃniens, summa est ethica; que veritatem docens incalunniabilem [sic] summa est logica.” 89. BAV Reg. lat. 1373, 108v–109r. Notes “theologiam, inquam, que causam causarum discutiens, summa est philosophia; que virtutum oﬃcia suis circumstantiis diﬃniens, summa est ethica; que veritatem docens incalunniabilem [sic] summa est logica.” 90. For the rankings, see the relevant entries in Farge, Biographical Register. 91. Moss, Renaissance Truth and the Latin Language Turn. 92. Farge, Orthodoxy and Reform, 33–37; Farge, Le Parti conservateur au XVIe siècle. Notes on contributor Christa Lundberg is Junior Research Fellow at St Catharine’s College in Cambridge. She has pub- lished an article about the philosophical letters of Charles de Bovelles and is working on a monograph about printing and the study of theology in early sixteenth-century Paris. Christa Lundberg http://orcid.org/0000-0002-5333-2995 Christa Lundberg http://orcid.org/0000-0002-5333-2995 Funding This work was supported by Arts and Humanities Research Council: [Grant Number AH/ L503897/1]. Disclosure statement No potential conﬂict of interest was reported by the author. 92. Farge, Orthodoxy and Reform, 33–37; Farge, Le Parti conservateur au XVIe siècle Manuscripts Anonymous. Paranymph Orations from 1514. Prague: Metropolitan chapter, ms. 832. De Lyon, Olivier. Paranymph Orations from 1510: Presentation Copy for Jean de Ganay. Rome: Biblioteca apostolica vaticana, ms. Reg. lat. 701. De Lyon, Olivier. Paranymph Orations from 1510: Presentation Copy for Jean de Ganay. Rome: Biblioteca apostolica vaticana, ms. Reg. lat. 1373. g Lasseré, Louis de. Paranymph Orations from 1512: Presentation Copy for Vaast Brioys. London: British Library, ms. Harley 2536. y y Lasseré, Louis de. Paranymph Orations from 1512. Paris: Bibliothèque nationale de France, ms. Latin 7812. Lasseré, Louis de. Paranymph Orations from 1512. Paris: Bibliothèque nationale de France, ms. Latin 7813. 16 C. LUNDBERG 16 Printed works Almain, Jacques. “Jacques Almain: Question at Vespers.” In Cambridge Translations of Renaissance Philosophical Texts, Vol. 2, edited by Jill Kraye, translated by Arthur Stephen McGrade, 13–35. Cambridge: University Press, 1997. Almain, Jacques. Libellus de auctoritate ecclesie, seu sacrorum conciliorum eam representationum … contra Thomam de Vio. Paris: J. Granjon, 1512. j Bakker, Paul J.J.M. “The Statutes of the College de Montaigu: Prelude to a Future Edition.” History of Universities Volume XXII/2, no. 22 (2008): 76. f Blair, Ann. “The Rise of Note-Taking in Early Modern Europe.” Intellectual History Review 20, no. 3 (September 2010): 303–316. doi:10.1080/17496977.2010.492611 Brinzei, M., and Christopher D. Schabel. “Les Cisterciens de l’université. Le cas du commentaire des Sentences de Conrad d’Ebrach (†1399).” In Les Cisterciens et la transmission des textes (XIIe- XVIIIe siècles), edited by Thomas Falmagne, Dominique Stutzmann, and Anne-Marie Turcan- Verkerk 453–486. Turnhout: Brepols, 2018. Broadie, Alexander. “John Mair’s Dialogus de Materia Theologo Tractanda: Introduction, Text, and Translation.” In Christian Humanism: Essays in Honour of Arjo Vanderjagt, edited by Alasdair A. MacDonald, Z.R.W.M. von Martel, and Jan R. Veenstra, 419–430. Leiden: Brill, 2009. Broadie, Alexander. The Circle of John Mair: Logic and Logicians in Pre-Reformation Scotland. Oxford: Clarendon, 1985. Budé, Guillaume. Opera omnia Vol. 1. Basel: N. Episcopus, 1557. Clerval, Alexandre. Registre des procès-verbaux de la Faculté de théologie de Paris, ‘1505-1523’. Paris: V. Lecoﬀre-J. Gabalda, 1917. Compère, Marie-Madeleine. “Montaigu.” In Les Collèges français 16e–18e siècle Répertoire 3 - Paris, edited by Marie-Madeleine Compère, 262–274. Paris: INRP, 2002. Compère, Marie-Madeleine. “Navarre.” In Les Collèges français 16e–18e siècle Répertoire 3 - Paris, edited by Marie-Madeleine Compère, 279–301. Paris: INRP, 2002. Du Boulay, César Égasse. Historia Universitatis Parisiensis … à Carolo M. ad nostra tempora ordine chronologico complectens. Vol. 6. Paris: F. Noel, 1673. Erasmus, Desiderius. In Opus epistolarum. Vol. 4, edited by P. S. Allen, and H. M. Allen. Oxford: Oxford University Press, 1922. Farge, James K. Biographical Register of Paris Doctors of Theology 1500–1536. Toronto: Pontiﬁcal Institute of Mediaeval Studies, 1980. Farge, James K. Le Parti conservateur au XVIe siècle : Université et Parlement de Paris à l’ép la Renaissance et de la Réforme. Paris: Collège de France, 1992. Farge, James K. “Noël Beda and the Defense of the Tradition.” In A Companion to Biblical Humanism and Scholasticism in the Age of Erasmus, edited by Erika Rummel, 143–164. Leiden: Brill, 2008. Farge, James K. Printed works Orthodoxy and Reform in Early Reformation France: The Faculty of Theology of Paris, 1500–1543. Leiden: Brill, 1985. Farge, James K. Students and Teachers at the University of Paris: The Generation of 1500. Leiden: Brill, 2006. Francus Vimacuus, Nicolas. Tres Hecatonomie de conceptibus. Paris: J. Barbier, 1509. Goulet, Robert. Compendium on the Magniﬁcence, Dignity, and Excellence of the University of Paris in the Year of Grace 1517, Translated by Robert Belle Burke. Philadelphia: University of Pennsylvania Press, 1928. Grandval, Marc de. Codex vesperiarum de optima politica tam ecclesiastica quam civili. Paris: J. Bade, 1513. Nathaël. Joannes Ravisius Textor (1492/3-1522): Un régent humaniste à Paris à l’aube de la aissance. Geneva: Droz, 2022. Istasse, Nathaël. “Pour une contribution à l’histoire de l’enseignement du latin à la Renaissance: les Progymnasmata primorum Navarriensis collegii grammaticorum Joannis Ravisii Textoris dis- cipulorum (manuscrit, 1516).” Camenae, no. 20 (2017): 1–48. 17 17 INTELLECTUAL HISTORY REVIEW 17 17 Kiss, Farkas Gábor. “‘O Pragensis Achademia!’ Ms. Prague, Metropolitan Chapter 832 and Its Relevance to the Eﬀorts of Church Uniﬁcation Between Hungary, Paris and Prague in 1518.” Archa Verbi. Yearbook for the Study Medieval Theology 9 (2012): 161–184. Kristeller, Paul Oskar. Medieval Aspects of Renaissance Learning. New York: Columbia University Press, 1992. noy, Jean. Regii Navarrae gymnasii Parisiensis historia. Vol. 1. Paris: Vidua E. Martini, 1677 Launoy, Jean. Regii Navarrae gymnasii Parisiensis historia. Vol. 1. Paris: Vidua E. Martini, 1677. 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https://openalex.org/W2213856969	https://hal.sorbonne-universite.fr/hal-01257840/document	English	null	The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria	Frontiers in microbiology	2,015	cc-by	12,222	To cite this version: Marjolaine Martin, Tristan Barbeyron, Renee Martin, Daniel Portetelle, Gurvan Michel, et al.. The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria. Frontiers in Microbiology, 2015, 6, pp.1487. 10.3389/fmicb.2015.01487. hal-01257840 The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria Marjolaine Martin, Tristan Barbeyron, Renee Martin, Daniel Portetelle, Gurvan Michel, Micheline Vandenbol The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria Marjolaine Martin, Tristan Barbeyron, Renee Martin, Daniel Portetelle, Gurvan Michel, Micheline Vandenbol Distributed under a Creative Commons Attribution 4.0 International License The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal- Polysaccharide-Degrading Bacteria Marjolaine Martin 1, Tristan Barbeyron 2, Renee Martin 1, Daniel Portetelle 1, Gurvan Michel 2 and Micheline Vandenbol 1 Marjolaine Martin 1, Tristan Barbeyron 2, Renee Martin 1, Daniel Portetelle 1, Gurvan Michel 2 and Micheline Vandenbol 1 1 Microbiology and Genomics Unit, Gembloux Agro-Bio Tech, University of Liège, Gembloux, Belgium, 2 Sorbonne Université, UPMC, Centre National de la Recherche Scientiﬁque, UMR 8227, Integrative Biology of Marine Models, Roscoff, France Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling. Yet the water column, which has been studied largely by metagenomic approaches, is poor in such bacteria and their algal-polysaccharide-degrading enzymes. Even more surprisingly, the few published studies on seaweed-associated microbiomes have revealed low abundances of such bacteria and their speciﬁc enzymes. However, as macroalgal cell-wall polysaccharides do not accumulate in nature, these bacteria and their unique polysaccharidases must not be that uncommon. We, therefore, looked at the polysaccharide-degrading activity of the cultivable bacterial subpopulation associated with Ascophyllum nodosum. From A. nodosum triplicates, 324 bacteria were isolated and taxonomically identiﬁed. Out of these isolates, 78 (∼25%) were found to act on at least one tested algal polysaccharide (agar, ι- or κ-carrageenan, or alginate). The isolates “active” on algal-polysaccharides belong to 11 genera: Cellulophaga, Maribacter, Algibacter, and Zobellia in the class Flavobacteriia (41) and Pseudoalteromonas, Vibrio, Cobetia, Shewanella, Colwellia, Marinomonas, and Paraglaceciola in the class Gammaproteobacteria (37). A major part represents likely novel species. Different proportions of bacterial phyla and classes were observed between the isolated cultivable subpopulation and the total microbial community previously identiﬁed on other brown algae. Here, Bacteroidetes and Gammaproteobacteria were found to be the most abundant and some phyla (as Planctomycetes and Cyanobacteria) frequently encountered on brown algae weren’t identiﬁed. At a lower taxonomic level, twelve genera, well-known to be associated with algae (with the exception for Colwellia), were consistently found on all three A. nosodum samples. Even more interesting, 9 of the 11 above mentioned genera containing polysaccharolytic isolates were predominant in this common core. The cultivable fraction of the bacterial community associated with A. nodosum is, thus, signiﬁcantly enriched in macroalgal-polysaccharide-degrading bacteria and these bacteria seem important for the seaweed holobiont even though they are under-represented in alga-associated microbiome studies. HAL Id: hal-01257840 https://hal.sorbonne-universite.fr/hal-01257840v1 Submitted on 18 Jan 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ORIGINAL RESEARCH published: 24 December 2015 doi: 10.3389/fmicb.2015.01487 ORIGINAL RESEARCH Keywords: Flavobacteriia, Gammaproteobacteria, macroalgae, agarase, carrageenase, alginate lyase, Ascophyllum nodosum, algal polysaccharidase Edited by: Olga Lage, University of Porto, Portugal Reviewed by: Torsten Thomas, The University of New South Wales, Australia Suhelen Egan, The University of New South Wales, Australia Correspondence: Marjolaine Martin marjolaine.martin@ulg.ac.be Correspondence: Marjolaine Martin marjolaine.martin@ulg.ac.be Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 29 October 2015 Accepted: 10 December 2015 Published: 24 December 2015 Received: 29 October 2015 Accepted: 10 December 2015 Published: 24 December 2015 INTRODUCTION As proof of feasibility, we used established activity tests and identiﬁed 13 esterases from Alphaprotobacteria and Gammaproteobacteria members, a GH3 β-glucosidase from an alphaprotobacterium, and a GH5 cellulase from a gammaproteobacterium (Martin et al., 2014a). Taking advantage of the ability of κ- and ι-carrageenans, agar, and alginate to form gels, we subsequently developed activity tests for screening a second A. nodusum metagenomic library for the corresponding polysaccharidases. The quality of this metagenomic library was validated and several esterases and β-glucosidases were again isolated. Surprisingly, however, we did not ﬁnd any MAPD enzymes (unpublished results). This disappointing result and the almost absence of MAPD f th t i t di d i t d Polysaccharide-degrading bacteria are key players in the global carbon cycle and have an increasing importance in biotechnology and biomass utilization. In terrestrial environments, plants host a remarkable diversity of microbes, representing a continuum of symbioses ranging from mutualism to commensalism to pathogenic behavior (Bulgarelli et al., 2013). Among these microbes, numerous bacteria possess polysaccharidases involved in the utilization of plant cell wall polymers (Gibson et al., 2011). Soils also constitute an important reservoir of hemicelluloytic microorganisms, which carry on the mineralization of plant polysaccharides (DeAngelis et al., 2010; Leung et al., 2015). In coastal ecosystems, macroalgae are crucial primary producers, constituting a huge biomass and playing an ecological role analogous to that of plants in terrestrial environments. Brown, green, and red macroalgae constitute three independent eukaryotic lineages, and their cell walls contain highly diverse polysaccharides, notably sulfated polysaccharides having no equivalent in land plants (Popper et al., 2011). By comparison with what is known about biomass recycling in terrestrial environments, knowledge of bacterial recycling of macroalgal biomass trails far behind. Nonetheless, the discovery of glycoside hydrolases (GH) and polysaccharide lyases (PL) targeting macroalgal polysaccharides has accelerated in the last 15 years (Michel and Czjzek, 2013). These particular enzymes were found to constitute either new subfamilies within known Carbohydrate- Active enZYmes (CAZy) families [e.g., κ-carrageenases and β-porphyranases in the GH16 family (Michel et al., 2001; Hehemann et al., 2010)] or new CAZy families, such as ι- carrageenases (GH82; Barbeyron et al., 2000), α-agarases (GH96; Flament et al., 2007), and fucoidanases (GH107; Colin et al., 2006). The majority of these enzymes have been isolated from seaweed-associated bacteria (Martin et al., 2014b). INTRODUCTION through activity screening of metagenomic libraries (Lee et al., 2010; Ferrer et al., 2015). Marine metagenomics has yielded disappointing results, however, as regards MAPD enzymes. For instance, in the entire Global Ocean Survey (GOS) dataset (Yooseph et al., 2007), there is not a single κ-carrageenase gene, only one ι-carrageenase gene, 30 coding DNA sequences (CDSs) for α-1,3-(3,6-anhydro)-L-galactosidase (the terminal enzyme of agar degradation, AhgA, GH117) and ﬁve times fewer CDSs for β-agarase and β-porphyranase, and seventeen PL17 alginate lyases (Hehemann et al., 2010; Rebuﬀet et al., 2011; Neumann et al., 2015). Clearly, this relative lack of success in ﬁnding MAPD enzymes in the water column suggests that bacterioplanktons rarely possess such enzymes. However, as most MAPD enzymes have been isolated from seaweed-associated bacteria, one should think that seaweed-associated microbiome datasets contain plenty of these speciﬁc enzymes. In fact, in the only two existing studies sequencing the microbiota associated with macroalgae (with Delisea pulchra and Ulva australis), no polysaccharidase gene is discussed (Fernandes et al., 2012), or only genes related to alginate metabolism are brieﬂy mentioned (Burke et al., 2011). Unfortunately, we couldn’t ﬁnd out if MAPD enzymes were present in the these macroalgae associated microbiota, as the metagenomic datasets of these two studies were deposited on the CAMERA database (http://camera.calit2.net/) that has shut down and its successor, iMicrobe (http://data.imicrobe.us/), does not contain these datasets. Also, a transcriptomic analysis of the microbiota of the red macroalga Laurencia dendroidea has been performed by RNA-Seq, but again, algal-polysaccharidase genes were either overlooked or absent from the dataset (de Oliveira et al., 2012). The metatranscriptomic datasets associated to L. dendroidea is publicly available on the MG-Rast database (de Oliveira et al., 2012). We searched in the protein ﬁles corresponding to these datasets sequence identities with the characterized MAPD enzymes (Michel and Czjzek, 2013; Martin et al., 2014b) using BlastP (NCBI), but we did not ﬁnd any homologous protein (data not shown). Concerning functional metagenomic analyses of seaweed-associated microbiota, only two studies have previously been carried out. On the one hand, the microbial community associated with Ulva australis was searched for antibacterial proteins (Yung et al., 2011). On the other hand, our group has recently screened the microbiota of the brown alga Ascophyllum nodosum for hydrolytic enzymes, especially glycoside hydrolases. Abbreviations: MAPD, Macroalgal-polysaccharide-degrading bacteria. Citation: Citation: Martin M, Barbeyron T, Martin R, Portetelle D, Michel G and Vandenbol M (2015) The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria. Front. Microbiol. 6:1487. doi: 10.3389/fmicb.2015.01487 December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 1 Cultivable Bacteria on Ascophyllum nodosum Martin et al. INTRODUCTION MAPD bacteria have been found essentially in two classes: (i) Gammaproteobacteria, in the phylum Proteobacteria (e.g., Agarivorans albus, Alteromonas agarilytica, Pseudoalteromonas carrageenovora, Vibrio sp. PO-303) and (ii) Flavobacteriia, in the phylum Bacteroidetes (e.g., Cellulophaga lytica, Flammeovirga yaeyamensis, Mariniﬂexile fucanivorans, Zobellia galactanivorans; for reviews see Michel and Czjzek, 2013; Martin et al., 2014b). Despite the identiﬁcation of these bacteria, most MAPD enzymes currently constitute very small protein families: e.g., GH16 κ-carrageenases (six proteins); GH16 β-agarases (41), GH16 β-porphyranases (7), GH82 ι-carrageenases (19), GH96 α-agarases (4), GH107 fucoidanases (4), and GH118 β-agarases (8) (http://www.cazy.org; Lombard et al., 2014). However, as macroalgal cell-wall polysaccharides do not accumulate in nature, MAPD bacteria and their unique polysaccharidases must not be that uncommon. The emerging metagenomic exploration of marine environments (Venter et al., 2004; Yooseph et al., 2007; Gómez-Pereira et al., 2012) oﬀers the promise of increasing the discovery of novel enzymes, either through data mining or This disappointing result and the almost absence of MAPD enzymes from other metagenomic studies on seaweed-associated microbiota raised several questions. Are MAPD bacteria December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 2 Cultivable Bacteria on Ascophyllum nodosum Martin et al. Supplementary Material (Table S1). A Principal Coordinates Analyses (PCoA) was realized with the relative abundance of each genus in each sample to represent the diﬀerences in composition of the three samples (Figure 1). Relative abundances of the genera were log transformed and Bray Curtis matrix distance was used. For each genus, a weight average was calculated based on the abundance of this genus in each sample and the axes-coordinates of the sample, in order to reveal aﬃnities between a genus and one, two or the three samples. The PCoA plot was performed using the VEGAN Package (Dixon, 2003; Oksanen et al., 2008) implemented in the R statistical software on the bacterial genera identiﬁed on the three algae samples (Figure 1). suﬃciently abundant at the surface of macroalgae? Are metagenomic analyses of entire seaweed-associated microbiota the most adequate approach to ﬁnding MAPD enzymes? As a ﬁrst step toward answering such questions, we have chosen to focus on the cultivable subpopulation of bacteria associated with A. nodosum and to establish a direct link with the catabolic capacities of these microorganisms. For this we have isolated, on a marine medium, 324 cultivable bacteria from triplicate A. nodosum samples. Isolation of Bacteria with Polysaccharidase Activities Phylogenetic and Statistical Analyses of the Bacterial Communities Isolated from the Three Ascophyllum nodosum Samples Healthy A. nodosum plants were collected in triplicate from the foreshore in Roscoﬀ(Brittany, France) at the end of March, 2014. The samples were rinsed three times with sterile seawater to remove loosely attached microorganisms and used immediately for microbial isolation. Cultivable microorganisms were isolated by swabbing algal surfaces with sterile cotton tips as in our previous functional metagenomics study (Martin et al., 2014a) and then inoculating plates of marine medium (Marine Agar, Difco). One plate per sample was inoculated and for each sample we used 20 cm of thallus. Plates were left at room temperature. After 4 days, 108 isolated colonies, representative of our total isolated colonies, were recovered at random from each plate (324 colonies in all) in 96-well plates with marine medium (Marine Broth, Difco). Glycerol (ﬁnal concentration 20%) was added to the liquid bacterial cultures in the 96-well plates and conserved at −80◦C. y The 324 isolated colonies were grown at 22◦C for 24 h in 96- microwell plates containing marine broth and then plated on marine broth containing 1.5% agar, 1% κ-carrageenan, 2% iota- carrageenan, or on modiﬁed Zobell medium (24.7 g/l NaCl, 6.3 g/l MgSO4, 0.7 g/l KCl, 5 g/l tryptone, 1 g/l yeast extract) with 5% alginate salts and 40 mM MgCl2. All chemicals were purchased from Sigma-Aldrich. After 48 h to 1 week, bacteria showing hydrolytic activity (a hole in the jelliﬁed medium for agarase or κ-carrageenase activity or complete liquefaction of the medium for ι-carrageenase or alginate lyase activity) were recovered and plated in order to obtain isolated colonies. Activity was conﬁrmed by testing again the isolated colonies for the observed activity/activities. INTRODUCTION We have taxonomically identiﬁed these bacterial isolates and have screened them for agarases, κ-carrageenases, ι-carrageenases, and alginate lyases. Bacterial isolates found to display at least one such activity were further investigated. 16s rRNA Sequence of the Bacterial Candidates Showing Polysaccharidase Activity y The 16S rRNA genes were ampliﬁed by PCR (Taq DNA polymerase, Roche) directly from the isolated colonies. Universal primers were used (8F, 5′-AGAGTTTGATCCTGGCTCAG- 3′ and 1492R, 3′-TACGGCTACCTTGTTACGACTT-5′). The ampliﬁed genes were sequenced by Sanger sequencing from both ends (forward and reverse; GATC Biotech, Germany). A consensus sequence was produced from the overlapping reads, for the 16S rRNA gene of each MAPD isolate. For each sequence, low quality 5 and 3′ extremities were manually trimmed, in order to keep only high quality sequences (DNA base peaks with quality scores above 30). Finally, the BLAST program (NCBI) was used to compare the obtained sequences against the GenBank database and closest neighbors were found by alignment with type strains with veriﬁed species names using EzTaxon (Kim et al., 2012). Sequences were manually aligned and curated with a representative set of 120 sequences of identiﬁed members of the family Flavobacteriaceae (Figure 2) and 355 of the class Gammaproteobacteria (Figure 3, Figure S1). Phylogenetic trees were constructed with these alignments using the Neighbor Joining Method (Saitou and Nei, 1987). Evolutionary distances were calculated according to Kimura’s two-parameter model using the gamma distribution (Kimura, 1980). To assign taxa to the 324 bacteria isolated from the three alga samples, the genus-speciﬁc V3-V4 region of the 16S rRNA (∼400 bp) was ampliﬁed and sequenced. First, 5 µl stock of bacteria in glycerol was mixed with 10 µl PCR water and heated at 95◦C for 10 min to lyse the bacteria. Then 5 µl bacterial lysate was added to the PCR mix [200 µM dNTP, 0.4 µM primer F (AllBactF 5′- TCCTACGGGAGGCAGCAGT-3′), 0.4 µM primer R (AllBactR 5′-GGACTACCAGGGTATCTAATCCTGTT-3′; Nadkarni et al., 2002; Stroobants et al., 2014), PCR buﬀer 1x, Taq DNA polymerase, ﬁnal volume 50 µl] for the PCR [94◦C- 7′, 35 × (95◦C-30′′), 60◦C-30′′, 65◦C -1′, 68◦C-7′]. Primers were produced at Eurogentec (BE) and PCR reagents were provided by Roche. PCR were realized in 96-well plates and amplicons were sequenced by Sanger Sequencing at GATC using the AllBactF forward primer (GATC Biotech, Germany). Sequences were aligned with sequences from the GenBank_Bacteria and SILVA SSU databases (E-value threshold 0.01) and each bacterium was taxonomically identiﬁed at the genus, family, order, class, and phylum level. Frontiers in Microbiology \| www.frontiersin.org 16s rRNA Sequence of the Bacterial Candidates Showing Polysaccharidase Activity Averages and percentage ranges of the resulting query coverages and identity percentages have been summarized for each sample and for the total isolated microbiota in the Frontiers in Microbiology \| www.frontiersin.org December 2015 \| Volume 6 \| Article 1487 3 Martin et al. Cultivable Bacteria on Ascophyllum nodosum FIGURE 1 \| PCoA of the genera found on the three alga samples. Bacterial classes are indicated in different colors. The size of each spot is proportional to the relative abundance of the genus indicated above or next to it. The closer a spot is to a sample name, the more abundant this genus is in that sample. The Genera common to just to samples are aligned along the gray arrows. The genera identiﬁed on the three samples (i.e., the common core) are in the gray shadowed circle. FIGURE 1 \| PCoA of the genera found on the three alga samples. Bacterial classes are indicated in different colors. The size of each spot is proportional to the relative abundance of the genus indicated above or next to it. The closer a spot is to a sample name, the more abundant this genus is in that sample. The Genera common to just to samples are aligned along the gray arrows. The genera identiﬁed on the three samples (i.e., the common core) are in the gray shadowed circle. Nucleotide Sequence Accession Numbers Sequence data of the V3-V4 region of the 16S rRNA gene of the non-MAPD isolates and the partial 16S rRNA of the 78 MAPD isolates were submitted to the EMBL database under accession numbers LN881131 to LN881422. recovered (108/sample) for further analysis. We succeeded in amplifying and sequencing the V3-V4 16S rRNA region from a total of 297 bacteria (98 from S1, 97 from S2, 102 from S3). These sequences were aligned to those from the GenBank and SILVA SSU databases (data not shown), with 291 out of 297 isolates being assigned to a genera. As a proof of quality sequence alignments, we had high average query coverage of 97.2% and identity percentage of 98.9% for the V3-V4 region sequences of these isolates (Table S1). For each A. nodosum sample, tables were made with the relative abundance and the frequency of each bacterial genus, family, order, class, and phylum identiﬁed (Table S2). Frontiers in Microbiology \| www.frontiersin.org FIGURE 2 \| Continued for review Martin et al., 2014b). Alphaproteobacteria emerged as a dominant phylum of the total bacterial community of these molecular studies (25–60%), whereas it represents only a minor fraction of the isolated cultivable fraction (11%). In contrast, the abundance of Bacteroidetes and Gammaproteobacteria members is two- to three-fold higher in our cultivable fraction. Surprisingly, no Planctomycetes and Cyanobacteria were isolated in our cultivable population even if they represent dominant phyla in the above mentioned reports of microbiota associated with other brown algae. Concerning the absence of Cyanobacteria, to our knowledge no representative of this autotrophic class has yet been identiﬁed by culturing methods dealing with algal microbiotas (Hollants et al., 2013). Furthermore, as our samples were taken in March 2014, a seasonal shift is likely to explain both the absence of Cyanobacteria and the high percentage of Gammaproteobacteria members among our isolates, as previously observed for F. vesiculosus (Lachnit et al., 2011). The absence of isolated Planctomycetes is more surprising, as these heterotrophic bacteria are expected to grow readily on complex culture media. for review Martin et al., 2014b). Alphaproteobacteria emerged as a dominant phylum of the total bacterial community of these molecular studies (25–60%), whereas it represents only a minor fraction of the isolated cultivable fraction (11%). In contrast, the abundance of Bacteroidetes and Gammaproteobacteria members is two- to three-fold higher in our cultivable fraction. Surprisingly, no Planctomycetes and Cyanobacteria were isolated in our cultivable population even if they represent dominant phyla in the above mentioned reports of microbiota associated with other brown algae. Concerning the absence of Cyanobacteria, to our knowledge no representative of this autotrophic class has yet been identiﬁed by culturing methods dealing with algal microbiotas (Hollants et al., 2013). Furthermore, as our samples were taken in March 2014, a seasonal shift is likely to explain both the absence of Cyanobacteria and the high percentage of Gammaproteobacteria members among our isolates, as previously observed for F. vesiculosus (Lachnit et al., 2011). The absence of isolated Planctomycetes is more surprising, as these heterotrophic bacteria are expected to grow readily on complex culture media. At lower taxonomic rank, molecular studies have provided evidence that total bacterial communities can diﬀer markedly from one individual alga to another and that the core community speciﬁc to the host is limited (∼15%; Burke et al., 2011; Bengtsson et al., 2012). Here, looking solely at the cultivable community of A. RESULTS AND DISCUSSION The Cultivable Microbiota of Ascophyllum nodosum: Comparison with the Total Bacterial Community of Brown Algae and Highlight of a Host-Speciﬁc Common Core A. nodosum is a large brown alga (up to 2 m) of the Fucaceae family which is common on both sides of North Atlantic ocean (Olsen et al., 2010). This macroalga is dominant along sheltered intertidal rocky shores and is a very long-lived species (10–15 years). The microbial population of A. nodosum was studied long ago by culturing (Chan and McManus, 1969) and electronic microscopy (Cundell et al., 1977). Surprisingly, these two old studies are the only ones concerning microorganisms associated with this brown seaweed. Cundell et al. (1977) describe the microbial epiphytes of A. nodosum as “a complex assemblage of end-attached bacteria, ﬁlamentous bacteria, ﬂexibacteria, yeasts, and pennate diatoms.” We here report the ﬁrst extensive characterization of the cultivable bacterial community of this ecologically relevant macroalga. In the total isolated bacterial population, 36 diﬀerent genera were identiﬁed. The most abundantly represented were Marinomonas (13.1%), Cellulophaga (10.1%), and Pseudoalteromonas (9.1%). Eighteen bacterial families were found, the major ones being Flavobacteriaceae (42.8%), Oceanospirillaceae (15.5%), and Rhodobacteraceae (11.1%). The most abundant phylum was Proteobacteria (56%, with class Alpha: 11.4%, Gamma: 44.1%, Beta: 0.7%), closely followed by Bacteroidetes (class Flavobacteriia 42.8%). Firmicutes (1.0%) members were identiﬁed only on sample S3 and constituted a very minor fraction of the cultivable community. Previous molecular studies on the kelp Laminaria hyperborean (Bengtsson and Øvreås, 2010; Bengtsson et al., 2012) and on Fucus vesiculosis (Lachnit et al., 2011; Stratil et al., 2013, 2014), closely related to A. nodosum, reveal the phyla- and class-level structures of their associated microbial communities to be markedly diﬀerent from the here identiﬁed population (see Approximately 3.103 bacteria were isolated from the surface of three A. nodosum thallus samples. A total of 324 bacteria were December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 4 Martin et al. Cultivable Bacteria on Ascophyllum nodosum FIGURE 2 \| Phylogenetic tree with the Flavobacteriaceae. 16S rRNA sequences of 120 representatives of the family Flavobacteriaceae were aligned with those of our 41 MAPD ﬂavobacterial isolates. Sequences (Continued) FIGURE 2 \| Continued were manually curated and phylogenetic trees were constructed with these alignments using the Neighbor Joining Method. Evolutionary distances were calculated according to Kimura’s two-parameter model using the gamma distribution (Kimura, 1980). FIGURE 2 \| Continued nodosum, we reach partly similar conclusions as there is some variability between the communities isolated on the three samples, as regards both the genera observed and their relative abundances (Figure 1, Table S2). On the PCoA plot (Figure 1), one can see that the samples are equally distant (dissimilar in their bacterial composition) from each other (Bray-Curtis distances between S1–S2 = 0.45; S1–S3 = 0.412; S2–S3 = 0.453). Moreover, some genera are speciﬁc to one sample (e.g., Hydrogenophaga, Ruegeria, and Tenacibaculum were found only on S1) or common to just two samples (e. g., Leucothrix, Psychroserpens, and Psychrobacter were identiﬁed only on S2 and S3). However, despite this genus diversity, 12 genera were consistently found on all three A. nosodum samples: (i) six Flavobacteriia genera, all of the Flavobacteriaceae family (Algibacter, Cellulophaga, Dokdonia, Formosa, Maribacter, and Zobellia); (ii) ﬁve Gammaproteobacteria genera; four genera of the order Alteromonadales but of diﬀerent families (Colwellia, Glaciecola, Pseudoalteromonas, and Shewanella) and one genus of the order Oceanospirillales (Marinomonas, Oceanospirillaceae family); and (iii) one Alphaproteobacteria genus (Sulﬁtobacter, Rhodobacteraceae family; Figure 1). These genera represent a core community of 33% of the cultivable subpopulation (12 out of 36 genera in total), which is twice the proportion observed in whole seaweed-associated microbiotas (Burke et al., FIGURE 2 \| Phylogenetic tree with the Flavobacteriaceae. 16S rRNA sequences of 120 representatives of the family Flavobacteriaceae were aligned with those of our 41 MAPD ﬂavobacterial isolates. Sequences (Continued) (Continued) December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 5 Martin et al. Cultivable Bacteria on Ascophyllum nodosum FIGURE 3 \| Phylogenetic tree with the Gammaproteobacteria. 16S rRNA sequence of 355 representatives of the class Gammaproteobacteria were aligned with those of our 37 MAPD Gammaproteobacterial isolates. Sequence were manually curated and phylogenetic trees were constructed with these alignments using the Neighbor Joining Method. Evolutionary distances were calculated according to Kimura’s two-parameter model using the gamma distribution (Kimura, 1980). FIGURE 3 \| Phylogenetic tree with the Gammaproteobacteria. 16S rRNA sequence of 355 representatives of the class Gammaproteobacteria were aligned with those of our 37 MAPD Gammaproteobacterial isolates. Sequence were manually curated and phylogenetic trees were constructed with these alignments using the Neighbor Joining Method. Evolutionary distances were calculated according to Kimura’s two-parameter model using the gamma distribution (Kimura, 1980). FIGURE 3 \| Phylogenetic tree with the Gammaproteobacteria. FIGURE 2 \| Continued 16S rRNA sequence of 355 representatives of the class Gammaproteobacteria were aligned with those of our 37 MAPD Gammaproteobacterial isolates. Sequence were manually curated and phylogenetic trees were constructed with these alignments using the Neighbor Joining Method. Evolutionary distances were calculated according to Kimura’s two-parameter model using the gamma distribution (Kimura, 1980). Frontiers in Microbiology \| www.frontiersin.org December 2015 \| Volume 6 \| Article 1487 Cultivable Bacteria on Ascophyllum nodosum Martin et al. 2011; Bengtsson et al., 2012). This common core includes several host-speciﬁc genera which are well known to associate with macroalgae. Cellulophaga, Pseudolateromonas, Shewanella, Sulﬁtobacter, and Zobellia, for example, are among the 33 genera isolated from all three macroalgal groups (brown, green, and red seaweeds), as determined on the basis of a review of 161 studies (mainly culture-based) dealing with bacteria associated with 159 macroalgal species (Hollants et al., 2013). Formosa, Dokdonia, Marinomonas, and Glaciecola have been also identiﬁed on other brown algae like Fucus vesiculosus (Lachnit et al., 2011; Goecke et al., 2013), F. evanescens (Ivanova et al., 2004a) or Laminaria sp. (Wiese et al., 2009; Dong et al., 2012). To our knowledge, Colwellia is the only genus retrieved here from our three alga samples (representing 3.4% of our total bacterial isolates) that has not previously been isolated from brown algae. The Colwelliaceae family (comprising the genera Colwellia and Thalassomonas) has already been identiﬁed on the red alga Delisea pulchra (Fernandes et al., 2012). Fernandes et al. found this family to be present on diseased thalli and absent from healthy thalli of this red alga. genus (Marinomonas) is represented here by only one MAPD isolate (with a single, alginolytic activity). Similarly, Formosa is the 6th most abundant genus, but none of the Formosa isolates displays any MAPD activity. This is particularly unexpected, since the characterized species Formosa agariphila is agarolytic (Nedashkovskaya, 2006) and its genome analysis has revealed a broad potential for degrading algal polysaccharides (Mann et al., 2013). This striking example is a reminder that extrapolating the biological or ecological function of a particular species to a larger bacterial group can lead to incorrect interpretations. Among the less abundant genera, Zobellia (13th, 10/297), Vibrio (15th, 7/297), and Shewanella (17th, 6/297) are also remarkable by their overrepresentation among the MAPD isolates (Figure 4B). Notably, 100% of the Zobellia isolates display MAPD activity. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: Novel Activities and Putative Novel Species The 324 isolated bacteria were plated and tested on culture media geliﬁed with agar, κ-carrageenan, ι-carrageenan, or alginate salts. We have found 78 isolates (24%) with polysaccharidase activity against at least one of the tested substrates (Table 1). Similar proportions of such bacteria were isolated from the three specimens collected (S1, 20%; S2, 23%; S3, 34%). Thus our study clearly indicates that the cultivable microbiota of A. nodosum is signiﬁcantly enriched in MAPD bacteria as compared to the total bacterial community (based on molecular studies and on to the diﬃculty to identify MAPD enzymes by functional genomics). Nonetheless, MAPD bacteria remain a minority even within the cultivable microbiota (24%). The entire 16S rRNA genes of the various candidates were ampliﬁed and sequenced. Sequences were aligned with those of type strains with valid species names (EzTaxon, Kim et al., 2012). The percentage of identity to the closest type strain and the polysaccharidase activities of each isolate are listed in Table 1. The ﬁrst remarkable result is that all the MAPD isolates belong to only two bacterial classes, despite the taxonomic diversity of the cultivable community: 41 isolates were identiﬁed as Flavobacteriia members and 37 as Gammaproteobacteria members (Table 1). It is also noteworthy that we have discovered MAPD activities in genera that were not previously known to include MAPD bacteria (Marinomonas and Colwellia strains) or to display the activities observed here (ι- and κ-carrageeenases in Algibacter and Maribacter isolates, respectively; Table 2). These novel activities may be explained by the likely isolation of novel MAPD species. Indeed, 63% of the MAPD isolates identiﬁed here have less than 98.65% sequence identity at 16S rRNA level to a known species (Figure 5). Therefore they could represent putative novel species even if there is still some discussion regarding the threshold percentage of 16S rRNA gene identity at which two species can be distinguished. A commonly accepted value is 97%. Recently, Kim et al. (2014), having compared the average nucleotide identities of almost 7000 prokaryotic genomes and their 16S rRNA gene identities, propose a threshold of 98.65%, while Tindall et al. (2010) stress that the 16S rRNA alone does not describe a species but only provides These isolates were assigned to 11 genera (out of the 36 genera isolated) and 8 families. Frontiers in Microbiology \| www.frontiersin.org FIGURE 2 \| Continued y y y Interestingly, the polysaccharidase activities identiﬁed here do not necessarily reﬂect the cell wall composition of A. nodosum. Brown alga cell walls are largely composed of alginates and sulfated fucoidans, whereas red macroalgae mainly contain sulfated galactans (agars or carrageenans; Popper et al., 2011). Unexpectedly, we have found similar proportions of bacterial isolates degrading polysaccharides of red (64/78) and brown (61/78) macroalgae (Figure 4B, Table 1), but these activities are not equally distributed between Gammaproteobacteria and Flavobacteriia. Most gammaproteobacterial isolates (26/37) exclusively degrade alginate (including all Cobetia, Marinomonas, and Vibrio isolates). All Pseudoalteromonas isolates are alginolytic, but a minority is also able to hydrolyze carrageenans (22%), and none of them is agarolytic. MAPD activities are more evenly distributed in Shewanella, all isolates being both alginolytic and carrageenolytic. More surprisingly, the Paraglaciecola and Colwellia isolates are not alginolytic, but can hydrolyze both agars and carrageenans. In contrast, the Flavobacteriia isolates are much more generalistic degraders. All the MAPD Flavobacteriia isolates hydrolyze red algal sulfated galactans (agars and/or carrageenans) and most of them (26/41) also alginate. This suggests that the MAPD Gammaproteobacteria isolates are more speciﬁc to brown algae, while the MAPD Flavobacteriia strains or species isolated from A. nodosum are likely to be found also on the surfaces of agarophytic and carrageenophytic red seaweeds. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: Among the ten most abundant genera in our total isolated population (Figure 4A), only four (Marinomonas, Cellulophaga, Pseudoalteromnas, and Maribacter) are represented by isolates with at least one polysaccharolytic activity. The genera Cellulophaga and Pseudoalteromonas are exceptional, being both abundant in the cultivable community (the second and third most abundant genera, respectively), and representing together more than 60% of the MAPD isolates (27/78 and 22/78, respectively; Figures 4A,B). In contrast, the most abundant December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 7 Cultivable Bacteria on Ascophyllum nodosum Cultivable Bacteria on Ascophyllum nodosum Martin et al. TABLE 1 \| Identiﬁed MAPD isolates, each with the closest type strain, the corresponding percentage of 16S rRNA gene similarity and the observed polysaccharidase activities. Isolated bacterial strain Closest bacterial strain (EZTaxon) % identity Polysaccharidase activitiesa Ag í-C κ-C AL ALGIBACTER sp. An67 Algibacter miyuki WS-MY6 97.75 + + – – CELLULOPHAGA sp. An8 Cellulophaga geojensis M-M6 97.88 + + + + An10 Cellulophaga geojensis M-M6 97.88 + + + + An42 Cellulophaga geojensis M-M6 97.66 + + + – An44 Cellulophaga geojensis M-M6 96.28 + + + + An47 Cellulophaga geojensis M-M6 96.6 + + + – An48 Cellulophaga geojensis M-M6 97.65 + + + – An11 Cellulophaga fucicola NN015860 95.56 + + + + An31 Cellulophaga fucicola NN015860 97.22 + + + + An38 Cellulophaga fucicola NN015860 97.51 + + + + An39 Cellulophaga fucicola NN015860 98 + + + + An40 Cellulophaga fucicola NN015860 96.75 + + + + An41 Cellulophaga fucicola NN015860 97.58 + + + + An43 Cellulophaga fucicola NN015860 96.83 + + + + An46 Cellulophaga fucicola NN015860 96.82 + + + – An49 Cellulophaga fucicola NN015860 97.65 + + + + An9 Cellulophaga baltica NN015840 98.27 + + + + An13 Cellulophaga baltica NN015840 98.64 + + + + An17 Cellulophaga baltica NN015840 97.75 + + + + An18 Cellulophaga baltica NN015840 98.49 + + + + An19 Cellulophaga baltica NN015840 98.20 + + + + An20 Cellulophaga baltica NN015840 97.45 + + + + An22 Cellulophaga baltica NN015840 95.87 + + + – An28 Cellulophaga baltica NN015840 96.32 + + + + An45 Cellulophaga baltica NN015840 98.63 + + + + An50 Cellulophaga baltica NN015840 97.68 + + + + An110 Cellulophaga baltica NN015840 98.53 + + + + An66 Cellulophaga paciﬁca KMM3664 96.21 + + + + MARIBACTER sp. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: An21 Maribacter aestuarii GY20 95.33 + + + + An70 Maribacter forsetii KT02ds 18-6 99.28 – + – + An72 Maribacter forsetii KT02ds 18-6 99.06 – + – + ZOBELLIA sp. An1 Zobellia galactanivorans DsiJ 99.4 + + + + An14 Zobellia galactanivorans DsiJ 97.63 + + – – An73 Zobellia galactanivorans DsiJ 97.7 + + – + An75 Zobellia galactanivorans DsiJ 98 + + – – An76 Zobellia galactanivorans DsiJ 97.8 + + – + An78 Zobellia galactanivorans DsiJ 97.5 + + – – An79 Zobellia galactanivorans DsiJ 97.8 + + – – An74 Zobellia laminariae KMM3676 97.6 + + – – An77 Zobellia laminariae KMM3676 96.8 + + – – An80 Zobellia laminariae KMM3676 98.9 + + + – (Continued) December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 8 Cultivable Bacteria on Ascophyllum nodosum Martin et al. Martin et al. Cultivable Bacteria on Ascophyllum nodosum TABLE 1 \| Continued Isolated bacterial strain Closest bacterial strain (EZTaxon) % identity Polysaccharidase activitiesa Ag í-C κ-C AL GAMMAPROTEOBACTERIA ALTEROMONADACEAE PARAGLACIECOLA sp. An27 Paraglaciecola mesophila KMM241 96.49 + + + – COLWELLIACEAE COLWELLIA sp. An23 Colwellia meonggei MA1-3 96.17 + + + – HALOMONODACEAE COBETIA sp. An107 Cobetia litoralis KMM 3880 99.31 – – – + An108 Cobetia litoralis KMM 3880 98.96 – – – + OCEANOSPIRILLACEAE MARINOMONAS sp. An109 Marinomonas ushuaiensis U1 97.14 – – – + PSEUDOALTEROMONODACEAE PSEUDOALTEROMONAS sp. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: An88 Pseudoalteromonas aliena KMM 3562 99.57 – – – + An89 Pseudoalteromonas aliena KMM 3562 99.2 – – – + An97 Pseudoalteromonas aliena KMM 3562 98.3 – – – + An33 Pseudoalteromonas espejiana NCIMB 2127 99.4 – + + + An51 Pseudoalteromonas espejiana NCIMB 2127 98.1 – – – + An81 Pseudoalteromonas espejiana NCIMB 2127 99.34 – – – + An82 Pseudoalteromonas espejiana NCIMB 2127 99.42 – – – + An83 Pseudoalteromonas espejiana NCIMB 2127 99.13 – – – + An84 Pseudoalteromonas espejiana NCIMB 2127 99.71 – + + + An85 Pseudoalteromonas espejiana NCIMB 2127 98.84 – – – + An86 Pseudoalteromonas espejiana NCIMB 2127 99.05 – – – + An87 Pseudoalteromonas espejiana NCIMB 2127 98.11 – – – + An90 Pseudoalteromonas espejiana NCIMB 2127 98.98 – + – + An93 Pseudoalteromonas espejiana NCIMB 2127 99.6 – – – + An94 Pseudoalteromonas espejiana NCIMB 2127 98.8 – – – + An95 Pseudoalteromonas espejiana NCIMB 2127 99.7 – – – + An99 Pseudoalteromonas espejiana NCIMB 2127 99.7 – + + + An100 Pseudoalteromonas espejiana NCIMB 2127 99.6 – + + + An101 Pseudoalteromonas espejiana NCIMB 2127 98.1 – – – + An106 Pseudoalteromonas espejiana NCIMB 2127 98.76 – – – + An96 Pseudolateromonas nigrifaciens NCIMB 8614 99.2 – – – + An98 Pseudolateromonas nigrifaciens NCIMB 8615 99.2 – – – + SHEWANELLACEAE SHEWANELLA sp. TABLE 1 \| Continued Isolated bacterial strain Closest bacterial strain (EZTaxon) % identity Polysaccharidase activitiesa Ag í-C κ-C AL GAMMAPROTEOBACTERIA ALTEROMONADACEAE PARAGLACIECOLA sp. An27 Paraglaciecola mesophila KMM241 96.49 + + + – COLWELLIACEAE COLWELLIA sp. An23 Colwellia meonggei MA1-3 96.17 + + + – HALOMONODACEAE COBETIA sp. An107 Cobetia litoralis KMM 3880 99.31 – – – + An108 Cobetia litoralis KMM 3880 98.96 – – – + OCEANOSPIRILLACEAE MARINOMONAS sp. An109 Marinomonas ushuaiensis U1 97.14 – – – + PSEUDOALTEROMONODACEAE PSEUDOALTEROMONAS sp. Frontiers in Microbiology \| www.frontiersin.org The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: FIGURE 4 \| (A) Percentage proportions of the most represented genera in the total isolated bacterial population ( ) and of the MAPD isolates belonging to these genera in the whole set of 78 MAPD isolates ( ); (B) Percentage proportions of MAPD isolates belonging to each MAPD-isolate-containing genus in the whole set of 78 MAPD isolates ( ), with their activities on red ( ) or brown seaweed galactans ( ). FIGURE 4 \| (A) Percentage proportions of the most represented genera in the total isolated bacterial population ( ) and of the MAPD isolates belonging to these genera in the whole set of 78 MAPD isolates ( ); (B) Percentage proportions of MAPD isolates belonging to each MAPD-isolate-containing genus in the whole set of 78 MAPD isolates ( ), with their activities on red ( ) or brown seaweed galactans ( ). a putative indication of a novel species Nevertheless, further phylogenetic tree, the Cellulophaga genus clearly appears non FIGURE 4 \| (A) Percentage proportions of the most represented genera in the total isolated bacterial population ( ) and of the MAPD isolates belonging to these genera in the whole set of 78 MAPD isolates ( ); (B) Percentage proportions of MAPD isolates belonging to each MAPD-isolate-containing genus in the whole set of 78 MAPD isolates ( ), with their activities on red ( ) or brown seaweed galactans ( ). FIGURE 4 \| (A) Percentage proportions of the most represented genera in the total isolated bacterial population ( ) and of the MAPD isolates belonging to these genera in the whole set of 78 MAPD isolates ( ); (B) Percentage proportions of MAPD isolates belonging to each MAPD-isolate-containing genus in the whole set of 78 MAPD isolates ( ), with their activities on red ( ) or brown seaweed galactans ( ). a putative indication of a novel species. Nevertheless, further taxonomic analyses and DNA-DNA hybridization experiments should be performed or average nucleotide identities determined to conﬁrm this (Tindall et al., 2010; Stackebrandt, 2011). The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: An88 Pseudoalteromonas aliena KMM 3562 99.57 – – – + An89 Pseudoalteromonas aliena KMM 3562 99.2 – – – + An97 Pseudoalteromonas aliena KMM 3562 98.3 – – – + An33 Pseudoalteromonas espejiana NCIMB 2127 99.4 – + + + An51 Pseudoalteromonas espejiana NCIMB 2127 98.1 – – – + An81 Pseudoalteromonas espejiana NCIMB 2127 99.34 – – – + An82 Pseudoalteromonas espejiana NCIMB 2127 99.42 – – – + An83 Pseudoalteromonas espejiana NCIMB 2127 99.13 – – – + An84 Pseudoalteromonas espejiana NCIMB 2127 99.71 – + + + An85 Pseudoalteromonas espejiana NCIMB 2127 98.84 – – – + An86 Pseudoalteromonas espejiana NCIMB 2127 99.05 – – – + An87 Pseudoalteromonas espejiana NCIMB 2127 98.11 – – – + An90 Pseudoalteromonas espejiana NCIMB 2127 98.98 – + – + An93 Pseudoalteromonas espejiana NCIMB 2127 99.6 – – – + An94 Pseudoalteromonas espejiana NCIMB 2127 98.8 – – – + An95 Pseudoalteromonas espejiana NCIMB 2127 99.7 – – – + An99 Pseudoalteromonas espejiana NCIMB 2127 99.7 – + + + An100 Pseudoalteromonas espejiana NCIMB 2127 99.6 – + + + An101 Pseudoalteromonas espejiana NCIMB 2127 98.1 – – – + An106 Pseudoalteromonas espejiana NCIMB 2127 98.76 – – – + An96 Pseudolateromonas nigrifaciens NCIMB 8614 99.2 – – – + An98 Pseudolateromonas nigrifaciens NCIMB 8615 99.2 – – – + SHEWANELLACEAE SHEWANELLA sp. An36 Shewanella paciﬁca KMM3597 98.3 – + + + An59 Shewanella paciﬁca KMM3597 97.6 – + + + An71 Shewanella paciﬁca KMM3597 97.6 – + + + An4 Shewanella japonica KMM 3299 98.3 – + – + VIBRIONACEAE VIBRIO sp. An91 Vibrio hemicentroti AlyHp32 99.17 – – – + An92 Vibrio splendidus ATCC 33125 97.2 – – – + An102 Vibrio splendidus ATCC 33125 98.99 – – – + An103 Vibrio splendidus ATCC 33125 99.14 – – – + An104 Vibrio splendidus ATCC 33125 99.21 – – – + An105 Vibrio splendidus ATCC 33125 99.28 – – – + 16S rRNA perecentage lower than 97% were indicated in red. aAg, agarase; í-C, ι-carrageenase; κ-C, κ-carrageenase; AL, alginate lyase. December 2015 \| Volume 6 \| Article 1487 9 Cultivable Bacteria on Ascophyllum nodosum Martin et al. Frontiers in Microbiology \| www.frontiersin.org The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: Indeed, 97% is the commonly accepted threshold percentage at which two species can be distinguished and 98.65% is the threshold proposed by Kim et al. (2014) which have compared the average nucleotide identities of almost 7000 prokaryotic genomes and their 16S rRNA gene identities. FIGURE 5 \| Ranges of 16S rRNA identity percentages for the identiﬁed MAPD isolates vs. known species. Two third of the MAPD isolates (<98.65% 16S rRNA identities) likely represent novel species. Indeed, 97% is the commonly accepted threshold percentage at which two species can be distinguished and 98.65% is the threshold proposed by Kim et al. (2014) which have compared the average nucleotide identities of almost 7000 prokaryotic genomes and their 16S rRNA gene identities. bleached thalli of the brown kelp Ecklonia radiata. They found Flavobacteriaceae and Oceanospirillaceae representatives to be more present on diseased tissues. Within these families, however, some genera were found in much higher proportion on healthy samples than on bleached ones, suggesting a role favorable to the macroalgal host. Interestingly, these genera include several of those represented by MAPD isolates obtained from A. nodosum: Zobellia, Maribacter, Pseudoalteromonas, Vibrio, Marinomonas, and Cobetia. Beyond their MAPD activities, species of these genera may have additional metabolic capacities advantageous for their hosts. This hypothesis is plausible at least for Zobellia species, which are known to synthesize an algal morphogenesis inducer (Matsuo et al., 2003). The role of MAPD bacteria within the total seaweed-associated microbiota is more obvious. These bacteria are essential for degrading intact cell-wall polysaccharides, and thus for releasing hydrolysis at the core group of cultivable bacteria. Indeed, MAPD activity was detected in 75% of the core genera (Algibacter, Cellulophaga, Colwellia, Glaciecola Maribacter, Marinomonas, Pseudoalteromonas, Shewanella and Zobellia; Figure 1, Table 1). Thus, even though MAPD bacteria constitute a minor fraction of both the total and cultivable bacterial communities, they apparently belong to the core group of bacteria living at the surface of A. nodosum and likely exert functions that are important for their macroalgal host and/or within the microbiota as a whole. How harboring MAPD bacteria might be beneﬁcial to the host is not obvious. Such bacteria have mostly been described as detrimental to macroalgae, being responsible for diseases, providing an entry for opportunistic bacteria, or accelerating algal degradation (Goecke et al., 2010; Egan et al., 2013; Hollants et al., 2013). Recently, Marzinelli et al. Frontiers in Microbiology \| www.frontiersin.org The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: However, to strengthen the taxonomic identiﬁcation of these MAPD isolates and the assumption that most of these isolates represent new species, phylogenetic trees of entire 16S rRNA genes were constructed for the Flavobactericeae (Figure 2) and Gammaproteobacteria members (Figure 3, Figure S1).The Flavobacteriaceae phylogenetic tree strongly suggests that we have identiﬁed three novel Zobellia species (represented by An80, An77, and the seven strains of the An14 clade) and a novel Maribacter species (An21; Figure 2). Furthermore, in this phylogenetic tree, the Cellulophaga genus clearly appears non- monophyletic as the MAPD Cellulophaga isolates are separated into two clades, 12 of them having a common ancestor with C. baltica and the other 16 a common ancestor with C. lytica. This result appears to conﬁrm the doubts raised in Bergey’s manual of Systematic Bacteriology concerning the monophyletic character of the Cellulopha genus (Krieg et al., 2011). In the detailed Gammaproteobacteria tree (Figure S1), the Colwellia sp. An23, the Paraglaciecola sp. An27, the four Shewanella isolated and the Marinomonas sp. An 109 seem very likely to represent novel species. Last but not least, one can observe that the proportion of MAPD bacteria increases dramatically while looking only Frontiers in Microbiology \| www.frontiersin.org December 2015 \| Volume 6 \| Article 1487 10 Cultivable Bacteria on Ascophyllum nodosum Martin et al. TABLE 2 \| Activities identiﬁed in our study and previously described for MAPD species or strains from the genera to which our 78 MAPD isolates were assigned. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: Genera to which the 78 Activities identiﬁed in our studya Previously described activitiesa References MAPD-isolates were assigned Ag í-C κ-C AL Ag í-C κ-C AL FLAVOBACTERIIA Algibacter • ■ • • Park et al., 2013; Tanaka et al., 2015 Cellulophaga • • • • • • • • Johansen et al., 1999; Park et al., 2012; Yao et al., 2013 Maribacter • • ■ • • • • Barbeyron et al., 2008 Zobellia • • • • • • • • Barbeyron et al., 2001; Nedashkovskaya et al., 2004 GAMMAPROTEOBACTERIA Paraglaciecola • • • • • • Romanenko et al., 2003; Yong et al., 2007 Colwellia ■ ■ ■ Browman, 2013; Liu et al., 2014; Wang et al., 2015 Cobetia • • Lelchat et al., 2015 Marinomonas ■ Macián et al., 2005; Lucas-Elió et al., 2011 Pseudolateromonas • • • • • • • Akagawa-Matsushita et al., 1992; Chi et al., 2014 Shewanella • • • • • • • Ivanova et al., 2001, 2003, 2004b; Wang et al., 2014 Vibrio • • • Sugano et al., 1993; Kim et al., 2013 • Activities found in our study and found previously for species/strains of this genus; ■Novel activities, that weren’t identiﬁed for any species/strains of this genus previously. aAg, agarase; í-C, ι-carrageenase; κ-C, κ-carrageenase; AL, alginate lyase. FIGURE 5 \| Ranges of 16S rRNA identity percentages for the identiﬁed MAPD isolates vs. known species. Two third of the MAPD isolates (<98.65% 16S rRNA identities) likely represent novel species. Indeed, 97% is the commonly accepted threshold percentage at which two species can be distinguished and 98.65% is the threshold proposed by Kim et al. (2014) which have compared the average nucleotide identities of almost 7000 prokaryotic genomes and their 16S rRNA gene identities. TABLE 2 \| Activities identiﬁed in our study and previously described for MAPD species or strains from the genera to which our 78 MAPD isolates were assigned. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: Genera to which the 78 Activities identiﬁed in our studya Previously described activitiesa References MAPD-isolates were assigned Ag í-C κ-C AL Ag í-C κ-C AL FLAVOBACTERIIA Algibacter • ■ • • Park et al., 2013; Tanaka et al., 2015 Cellulophaga • • • • • • • • Johansen et al., 1999; Park et al., 2012; Yao et al., 2013 Maribacter • • ■ • • • • Barbeyron et al., 2008 Zobellia • • • • • • • • Barbeyron et al., 2001; Nedashkovskaya et al., 2004 GAMMAPROTEOBACTERIA Paraglaciecola • • • • • • Romanenko et al., 2003; Yong et al., 2007 Colwellia ■ ■ ■ Browman, 2013; Liu et al., 2014; Wang et al., 2015 Cobetia • • Lelchat et al., 2015 Marinomonas ■ Macián et al., 2005; Lucas-Elió et al., 2011 Pseudolateromonas • • • • • • • Akagawa-Matsushita et al., 1992; Chi et al., 2014 Shewanella • • • • • • • Ivanova et al., 2001, 2003, 2004b; Wang et al., 2014 Vibrio • • • Sugano et al., 1993; Kim et al., 2013 • Activities found in our study and found previously for species/strains of this genus; ■Novel activities, that weren’t identiﬁed for any species/strains of this genus previously. aAg, í C C AL l i t l TABLE 2 \| Activities identiﬁed in our study and previously described for MAPD species or strains from the genera to which our 78 MAPD isolates were assigned r study and previously described for MAPD species or strains from the genera to which our 78 MAPD isolates were TABLE 2 \| Activities identiﬁed in our study and previously described for MAPD species or strains from the genera to which our 78 MAPD isolates were assigned. us; ■Novel activities, that weren’t identiﬁed for any species/strains of this genus previously. aAg, ■Novel activities, that weren’t identiﬁed for any species/strains of this genus previously. aAg, • Activities found in our study and found previously for species/strains of this genus; ■Novel activities, that weren’t identiﬁed for any species/strains of this genus previously. aAg, agarase; í-C, ι-carrageenase; κ-C, κ-carrageenase; AL, alginate lyase. FIGURE 5 \| Ranges of 16S rRNA identity percentages for the identiﬁed MAPD isolates vs. known species. Two third of the MAPD isolates (<98.65% 16S rRNA identities) likely represent novel species. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: Genera to which the 78 Activities identiﬁed in our studya Previously described activitiesa References MAPD-isolates were assigned Ag í-C κ-C AL Ag í-C κ-C AL FLAVOBACTERIIA Algibacter • ■ • • Park et al., 2013; Tanaka et al., 2015 Cellulophaga • • • • • • • • Johansen et al., 1999; Park et al., 2012; Yao et al., 2013 Maribacter • • ■ • • • • Barbeyron et al., 2008 Zobellia • • • • • • • • Barbeyron et al., 2001; Nedashkovskaya et al., 2004 GAMMAPROTEOBACTERIA Paraglaciecola • • • • • • Romanenko et al., 2003; Yong et al., 2007 Colwellia ■ ■ ■ Browman, 2013; Liu et al., 2014; Wang et al., 2015 Cobetia • • Lelchat et al., 2015 Marinomonas ■ Macián et al., 2005; Lucas-Elió et al., 2011 Pseudolateromonas • • • • • • • Akagawa-Matsushita et al., 1992; Chi et al., 2014 Shewanella • • • • • • • Ivanova et al., 2001, 2003, 2004b; Wang et al., 2014 Vibrio • • • Sugano et al., 1993; Kim et al., 2013 • Activities found in our study and found previously for species/strains of this genus; ■Novel activities, that weren’t identiﬁed for any species/strains of this genus previously. aAg, agarase; í-C, ι-carrageenase; κ-C, κ-carrageenase; AL, alginate lyase. TABLE 2 \| Activities identiﬁed in our study and previously described for MAPD species or strains from the genera to which our 78 MAPD isolates were assigned. REFERENCES the kelp Laminaria hyperborea. ISME J. 6, 2188–2198. doi: 10.1038/ismej. 2012.67 the kelp Laminaria hyperborea. ISME J. 6, 2188–2198. doi: 10.1038/ismej. 2012.67 Akagawa-Matsushita, M., Matsuo, M., Koga, Y., and Yamasato, K. (1992). Alteromonas atlantica sp. nov. and Alteromonas carrageenovora sp. nov., bacteria that decompose algal polysaccharides. Int. J. Syst. Bacteriol. 42, 621–627. doi: 10.1099/00207713-42-4-621 Browman, J. P. (2013). “The family colwelliaceae,” in The Prokaryotes: Gammaproteobacteria, eds E. Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt, and F. Thompson (Berlin; Heidelberg: Springer), 179–195. Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., and Schulze- Lefert, P. (2013). Structure and functions of the bacterial microbiota of plants. Annu. Rev. Plant Biol. 64, 807–838. doi: 10.1146/annurev-arplant-050312- 120106 Barbeyron, T., Carpentier, F., L’Haridon, S., Schüler, M., Michel, G., and Amann, R. (2008). Description of maribacter forsetii sp. nov., a marine Flavobacteriaceae isolated from North Sea water, and emended description of the genus Maribacter. Int. J. Syst. Evol. Microbiol. 58, 790–797. doi: 10.1099/ijs.0.65469-0 Burke, C., Thomas, T., Lewis, M., Steinberg, P., and Kjelleberg, S. (2011). Composition, uniqueness and variability of the epiphytic bacterial community of the green alga Ulva australis. ISME J. 5, 590–600. doi: 10.1038/ismej. 2010.164 Barbeyron, T., L’Haridon, S., Corre, E., Kloareg, B., and Potin, P. (2001). Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from a red alga, and classiﬁcation of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov. Int. J. Syst. Evol. 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BMC Microbiol. 10:261. doi: 10.1186/1471-2180-10-261 Appl. Biochem. Biotechnol. 173, 1703–1716. doi: 10.1007/s12010-014-0958-3 Colin, S., Deniaud, E., Jam, M., Descamps, V., Chevolot, Y., Kervarec, N., et al. (2006). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01487 Table S1 \| Percentage ranges and averages of the query coverages and identity percentages for the alignments with the 291 V3-V4 region sequences of the isolates to which a genus have been attributed. Table S2 \| Abundance (#) and percentage proportion of each genus, family, order, class, and phylum identiﬁed on each sample. Figure S1 \| Detailed phylogenetic tree for the Gammaproteobacteria. The Cultivable Microbiota from A. nodosum is Enriched in MAPD Bacteria: (2015) compared microbial communities on healthy and December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 11 Cultivable Bacteria on Ascophyllum nodosum Martin et al. ACKNOWLEDGMENTS We thank Michèle Nuttinck (B) and Murielle Jam (FR) for their help with sample collection and treatment and Florine Degrune and Marc Dufrêne (B) for their help with the PCoA. CONCLUSION In terrestrial environments, the bacteria involved in recycling plant polysaccharides are essentially found both on living plants and in the soils that immediately surround them. The situation is more complex for marine macroalgae. They live attached to rocks, and when algal fragments are released, they are quickly dispersed by the waves and tides. The available marine metagenomic data show that the water column is a habitat poor in MAPD bacteria and, for a macroalga, not equivalent to a surrounding soil. Tidal sediments could be crucial reservoirs of MAPD bacteria, but this remains an open question. A third environment likely to be a habitat for MAPD bacteria is the surface of the macroalgae themselves. We have shown here that this is indeed the case and that the cultivable microbiota of healthy A. nodosum specimens is enriched in MAPD bacteria. These bacteria, however, are not the most abundant ones associated with brown seaweeds; they constitute a minority fraction even within the cultivable subpopulation. An attractive hypothesis is that this low abundance of MAPD bacteria is due to active and/or passive defense systems of the macroalga, preventing proliferation of these potentially harmful bacteria. Evidence of such defense systems in macroalgae has been accumulating over the last decade (Potin et al., 2002; Egan et al., 2014). If this hypothesis is correct, one can expect MAPD bacteria to bloom on weakened or dead macroalgae, thus contributing signiﬁcantly to recycling of macroalgal biomass. As regards bioprospecting, our work demonstrates that culturing (combined, for instance, with subsequent genome sequencing of cultivable isolates) is an eﬃcient strategy for ﬁnding new MAPD bacteria and their corresponding polysaccharidases. FUNDING This project was funded by Gembloux Agro-Bio Tech (ULg), Wallonie-Bruxelles International (WBI), and the Fonds Scientiﬁque de la Recherche (F.R.S-F.N.R.S) in the framework of the Collaboration Program Hubert Curien. GM and TB are grateful for support by the French Government through the National Research Agency with regard to the “Blue Enzymes” ANR project with reference ANR-14-CE19-0020-01. AUTHOR CONTRIBUTIONS products assimilable by the much more abundant bacteria (e.g., Alphaproteobacteria) lacking these unique MAPD enzymes. products assimilable by the much more abundant bacteria (e.g., Alphaproteobacteria) lacking these unique MAPD enzymes. Conceived and designed the experiments: MM, MV, GM, TB, DP. Performed the experiments: MM, RM. Analyzed the data: MM, MV, GM, TB. Contributed reagents/materials/analysis tools: MM, RM. Wrote the manuscript: MM, GM. Conceived and designed the experiments: MM, MV, GM, TB, DP. Performed the experiments: MM, RM. Analyzed the data: MM, MV, GM, TB. Contributed reagents/materials/analysis tools: MM, RM. Wrote the manuscript: MM, GM. REFERENCES Introducing EzTaxon-e: a prokaryotic 16s rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62, 716–721. doi: 10.1099/ijs.0.038075-0 Egan, S., Fernandes, N. D., Kumar, V., Gardiner, M., and Thomas, T. (2014). 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Microbiol. 13, 1253–1270. doi: 10.1111/j.1462- 2920.2011.02426.x Copyright © 2015 Martin, Barbeyron, Martin, Portetelle, Michel and Vandenbol. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Romanenko, L. A., Zhukova, N. V., Rohde, M., Lysenko, A. M., Mikhailov, V. V., and Stackebrandt, E. (2003). Glaciecola mesophila sp. nov., a novel marine agar-digesting bacterium. Int. J. Syst. Evol. Microbiol. 53, 647–651. doi: 10.1099/ijs.0.02469-0 December 2015 \| Volume 6 \| Article 1487 Frontiers in Microbiology \| www.frontiersin.org 14
https://openalex.org/W2904776564	https://www.mdpi.com/2226-4787/7/1/3/pdf?version=1545732694	English	null	Risk of Transverse Myelitis Following Dengue Infection: A Systematic Review of the Literature	Pharmacy	2,018	cc-by	5,991	Received: 17 November 2018; Accepted: 19 December 2018; Published: 23 December 2018 Abstract: Introduction: Dengue virus (DENV) is one of the most common arbovirus diseases, with a wide spectrum of presentation. Spinal cord involvement in dengue infection (DF) is rare. However, the risk of transverse myelitis (TM) following Dengue has not been systematically assessed. Methods: We undertook a systematic review of published English literature from January 1974 to December 2017 to assess risk of TM and outcomes following DF. Data sources included EMBASE, MEDLINE, Cochrane library, ISI web of knowledge, conference proceedings and references within identiﬁed articles. Results: We identiﬁed 242 potential studies, 62 were duplicates. A further 136 were excluded on the basis of title and abstract and 19 studies did not meet the eligibility criteria on full text screening. We included 25 publications involving 2672 cases of DF. A small proportion (10.8%, (289/2672)) had neurological complications, of which 2.3% (61/2672) was TM. For articles reporting epidemiological data, the neurological complication was twice in males compared to female 67.7% (130/192) vs. 32.7% (62/192) and 1.5-fold increase TM for males 59.3% (32/54) vs 40.7% (22/54). The mean age at presentation was 33.1years (range 0.75–61), with onset at 11.7days. The method of diagnosing TM due to DF was mainly IgM seropositivity 92% (n = 23/25) and the commonest treatment modality was steroid 78.3% (n = 18/23). Only half had full recovery 50.8% (n = 31/61). There was no mortality following dengue, however, the crude case fatality rate following TM was 3.3% (n = 2/61). Conclusion: This review highlights the risk of TM following dengue. Although neurological complications are rare, especially TM, once set in, it is associated with a signiﬁcant morbidity. Keywords: Dengue Fever; transverse myelitis; risk; systematic review pharmacy pharmacy Risk of Transverse Myelitis Following Dengue Infection: A Systematic Review of the Literature 1 School of Medicine, Imperial College London, London SW7 2AZ, UK; naﬁsa.badat13@imperial.ac.uk (N.B.) da1813@ic.ac.uk (D.A.) 1 School of Medicine, Imperial College London, London SW7 2AZ, UK; naﬁsa.badat13@imperial.ac.uk (N.B. da1813@ic.ac.uk (D.A.) 2 Department of Family Medicine, University of Benin Teaching Hospital (UBTH), Benin City, Nigeria; petolis2008@gmail.com 3 Department of Medicine, Queen’s Hospital, Romford RM7 0AG, UK; olakunle.ojubolamo1@nhs.net 4 Paediatric Infectious Diseases Research Group, Institute for Infection and Immunity, St. George’s, University of London, London SW17 0RE, UK 3 Department of Medicine, Queen’s Hospital, Romford RM7 0AG, UK; olakunle.ojubolamo1@nhs.net 4 Paediatric Infectious Diseases Research Group, Institute for Infection and Immunity, St. George’s, University of London, London SW17 0RE, UK * Correspondence: godwin.oligbu@nhs.net * Correspondence: godwin.oligbu@nhs.net Received: 17 November 2018; Accepted: 19 December 2018; Published: 23 December 2018 pharmacy Review Risk of Transverse Myelitis Following Dengue Infection: A Systematic Review of the Literature Naﬁsa Badat 1, Dalia Abdulhussein 1, Peter Oligbu 2, Olakunle Ojubolamo 3 and Godwin Oligbu 4,* 1 School of Medicine, Imperial College London, London SW7 2AZ, UK; naﬁsa.badat13@imperial.ac.uk (N.B.); da1813@ic.ac.uk (D.A.) 2 Department of Family Medicine, University of Benin Teaching Hospital (UBTH), Benin City, Nigeria; petolis2008@gmail.com 3 Department of Medicine, Queen’s Hospital, Romford RM7 0AG, UK; olakunle.ojubolamo1@nhs.net 4 Paediatric Infectious Diseases Research Group, Institute for Infection and Immunity, St. George’s, University of London, London SW17 0RE, UK * Correspondence: godwin.oligbu@nhs.net Received: 17 November 2018; Accepted: 19 December 2018; Published: 23 December 2018 pharmacy Review Risk of Transverse Myelitis Following Dengue Infection: A Systematic Review of the Literature Naﬁsa Badat 1, Dalia Abdulhussein 1, Peter Oligbu 2, Olakunle Ojubolamo 3 and Godwin Oligbu 4,* 1 School of Medicine, Imperial College London, London SW7 2AZ, UK; naﬁsa.badat13@imperial.ac.uk (N.B.); da1813@ic.ac.uk (D.A.) 2 Department of Family Medicine, University of Benin Teaching Hospital (UBTH), Benin City, Nigeria; petolis2008@gmail.com 3 Department of Medicine, Queen’s Hospital, Romford RM7 0AG, UK; olakunle.ojubolamo1@nhs.net 4 Paediatric Infectious Diseases Research Group, Institute for Infection and Immunity, St. George’s, University of London, London SW17 0RE, UK * Correspondence: godwin.oligbu@nhs.net Received: 17 November 2018; Accepted: 19 December 2018; Published: 23 December 2018 2.2. Study Selection Studies were eligible for inclusion if they reported neurological complications following dengue infection which were relevant to our study focus; to review the risk of TM following dengue infection. Articles irrelevant to the study were excluded or if they didn’t mention the risk of TM in relation to dengue infection. Two reviewers (G.O. and N.B.) independently screened the titles and abstracts of papers identiﬁed by the electronic searches, evaluating exclusion and inclusion criteria for all papers. We retrieved full text articles of included publications and each was then independently reviewed for eligibility. 2.1. Data Search and Selection A search was designed to identify case reports and observational studies (case series, cohort study, case–control study) reporting transverse myelitis as a complication of dengue viral infection. It aimed to include all publications that evaluated the current data in use of the risk of transverse myelitis (TM) following dengue virus infection globally. We searched MEDLINE, ISI web of knowledge, conference proceedings and EMBASE from 1 January 1974 to 26 December 2017. Both free text and the use of medical subheadings (MeSH) terms were used as search items. An initial search was conducted in order to scope all appropriate search terms followed by a more extensive search using two similar search criteria. The MeSH terms and free text terms used are included in the Appendix A. Studies were excluded if they were individual opinion or non-availability of full text, experimental or laboratory studies or not original research. We only included studies published in English language in our review. After the initial screening process, all publications were assessed for eligibility based on their titles followed by abstracts and full text. 1. Introduction Dengue is a viral disease transmitted by the Aedes mosquito and is endemic in tropical and subtropical areas, in particular the Americas and Asia. This puts an estimated 4 billion people at risk of acquiring the virus; currently it is estimated 100 million cases of symptomatic dengue occur annually [1]. Lack of treatment and immunisation therapy, as well as inadequate vector control have meant that there are no options in the management of severe disease apart from supportive measures [1,2]. In addition, with population growth and increased intercontinental travel over the past decade, it is more likely, if no other combative measures are utilised, that the number of cases will continue to increase. Pharmacy 2019, 7, 3; doi:10.3390/pharmacy7010003 www.mdpi.com/journal/pharmacy www.mdpi.com/journal/pharmacy 2 of 11 Pharmacy 2019, 7, 3 Pharmacy 2019, 7, 3 Pharmacy 2019, 7, 3 The vast majority of cases are asymptomatic [1], and where symptoms do occur, these commonly manifest with a fever, generalised pain, nausea and vomiting [2,3]. Severity of infection has been traditionally assessed by cardiovascular compromise, but most recently is the addition of central nervous system (CNS) involvement as a factor of severity since the number of cases describing dengue neurotropism have come to light [2,3]. This may be because factors contributing to neurological manifestations are themselves of increased severity of disease, for example prolonged shock, hepatic failure and hyponatraemia [4–6]. Damage to the spinal cord (myelitis) following infection can occur during infection (parainfectious) via direct invasion, or after infection (postinfectious) via a proposed immune-mediated inﬂammatory process [3,7,8]. Transverse myelitis has been described in a number of case reports where the main manifestations are sensorimotor disturbance of the lower limbs and urinary retention [7,9–15]. Currently the mechanisms of spinal cord damage in dengue are poorly deﬁned and the exact burden of these neurological manifestations is yet to be fully assessed. Hence, we summarised the literature on the risks of transverse myelitis following dengue infection as well as the proposed mechanisms behind this. 2.3. Quality Assessment and Data Extraction Two independent reviewers (G.O. and N.B.) reviewed the methodological quality of included studies, the comparability of case and controls, and outcomes. The explanatory variables extracted included: country, study design, description of study subjects, underlying comorbidity, clinical presentation, management and the outcome of the patient with TM. The study quality assessment was undertaken using the Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement for the conduct and reporting of systematic reviews [16]. 3 of 11 Pharmacy 2019, 7, 3 2.4. Data Analysis Pharmacy 2018, 6, x FOR Eligible studies were summarised using descriptive analyses to provide the overview of the information on populations studied, clinical presentations, underlying comorbidity and patient mortality outcomes. We calculated the age and sex distribution of TM generalised from the extracted data. We also calculated the risk of TM following dengue infection in children following dengue infection and compared this with that obtained in the adults for the outcome of interest where data were available. We calculated the crude fatality rate as the total number of mortality following TM divided by total number of reported TM cases over the same period. Eligible studies were then analysed qualitatively using Microsoft Ofﬁce Excel 2007 and summarised. 2.4. Data Analysis Eligible studies were summarised using descriptive analyses to provide the overview of the information on populations studied, clinical presentations, underlying comorbidity and patient mortality outcomes. We calculated the age and sex distribution of TM generalised from the extracted data. We also calculated the risk of TM following dengue infection in children following dengue infection and compared this with that obtained in the adults for the outcome of interest where data were available. We calculated the crude fatality rate as the total number of mortality following TM divided by total number of reported TM cases over the same period. Eligible studies were then analysed qualitatively using Microsoft Office Excel 2007 and summarised. 3. Results 3. Results We identiﬁed 242 potential studies, of which 62 were duplicates. A further 136 were excluded on the basis of title and abstract and 19 studies did not meet the eligibility criteria on full text screening (Figure 1). The remaining 25 studies were eligible and full text was assessed for inclusion in the ﬁnal review [7,9,10,12–15,17–34]. Most of the studies were from Asia (76%; 19/25) and the rest from South America (24%; 6/25). The majority of the studies were case reports (64%; 16/25), case series (8%; 2/25), cohort study (16%; 4/25), cross-sectional studies (8%; 2/25) and one prospective study (4%; 1/25). Only ﬁve studies reported dengue serotype; three studies had serotype 1 only, serotype 2 only, and serotype 3 only. The other two studies displayed had either dengue serotypes 1–3, or all four serotypes. A summary of the study design, study subjects, data collection method, and treatment is presented in Tables 1 and 2. Most of the included studies did not report the ethnicity. We identified 242 potential studies, of which 62 were duplicates. A further 136 were excluded on the basis of title and abstract and 19 studies did not meet the eligibility criteria on full text screening (Figure 1). The remaining 25 studies were eligible and full text was assessed for inclusion in the final review [7,9,10,12–15,17–34]. Most of the studies were from Asia (76%; 19/25) and the rest from South America (24%; 6/25). The majority of the studies were case reports (64%; 16/25), case series (8%; 2/25), cohort study (16%; 4/25), cross-sectional studies (8%; 2/25) and one prospective study (4%; 1/25). Only five studies reported dengue serotype; three studies had serotype 1 only, serotype 2 only, and serotype 3 only. The other two studies displayed had either dengue serotypes 1–3, or all four serotypes. A summary of the study design, study subjects, data collection method, and treatment is presented in Tables 1 and 2. Most of the included studies did not report the ethnicity. Figure 1. Identification and the selection of eligible studies in the systematic review. Figure 1. Identiﬁcation and the selection of eligible studies in the systematic review. Figure 1. Identification and the selection of eligible studies in the systematic review. Figure 1. Identiﬁcation and the selection of eligible studies in the systematic review. 4 of 11 Pharmacy 2019, 7, 3 Table 1. Description of the study design and the reported outcomes. 3. Results 3. Results Study Year of Study Country Study Design Number of Dengue Cases Diagnosis Method Outcome Singh et al. [17] 2013 India Case Report 1 Dengue IgM seropositivity 1 patient died. Ghosh et al. [18] 2011 India Case Report 1 Dengue IgM seropositivity CSF analysis full recovery Seet et al. [7] 2006 Singapore Case Report 1 Antibody index ratio of dengue IgM:IgG full recovery Kunishige et al. [13] 2004 Singapore Case Report 1 Dengue IgM seropositivity (CSF) partial recovery Fong CY et al. [19] 2016 Malaysia Case Report 1 Dengue IgM seropositivity full recovery Gupta et al. [20] 2013 India Case report 1 Dengue IgM seropositivity (ELISA) History NS1 ag assay full recovery Wasay et al. [21] 2008 Pakistan Case Series 6 Dengue IgM seropositivity 4 patients made a full recovery, 2 patients died. Samanta et al. [22] 2012 India Case Series 3 Dengue IgM seropositivity (serum/viral/blood) 1 patient made a full recovery, 1 patient made a partial recovery, 1 patient died. Misra et al. [23] 2015 India Case Study 116 Dengue IgM seropositivity History, Exam, NS1 antigen assay 78 patients made a full recovery, 27 patients made a partial recovery, 11 patients died. Sahu et al. [24] 2014 India Cohort 484 Dengue IgM seropositivity 479 patients made a full recovery, 5 patients died Soars et al. [25] 2006 Brazil Cross-sectional study 13 Dengue IgM seropositivity (blood/CSF (ELISA)) 12 patients made a full recovery, 1 patient with encephalitis died Weeratunga et al. [26] 2014 Sri Lanka Cross-sectional Study 7 Dengue IgM seropositivity (blood/CSF) 6 patients made a full recovery, 1 patient made a partial recovery. Puccioni-Sohler et al. (Brazil) [12] 2009 Brazil Retrospective study 27 Dengue IgM seropositivity Partial recovery Larik et al. [10] 2012 Singapore Case Report 1 Dengue IgM seropositivity Dengue RNA Full recovery Lim et al. [27] 2012 Singapore Case Report 1 Dengue IgM seropositivity Partial recovery Tomar et al. [28] 2015 India Case Report 1 Dengue IgM seropositivity Full recovery Mo et al. [29] 2016 China Case Report 1 Dengue IgM/IgG seropositivity (CSF) Partial recovery Mota et al. [30] 2017 Brazil Case Report 1 Dengue IgM seropositivity Partial recovery Leão et al. [14] 2000 Brazil Case Report 1 Dengue IgM seropositivity (CSF) Full recovery Miranda de Sousa A et al. [31] 2014 Brazil Case Report 1 Dengue IgM seropositivity (CSF) Full recovery Renganathan et al. Note: Abbreviations: IV: intravenous; IgM: immunoglobulin M; IgG: immunoglobulin G; NA: not available; CSF: cerebrospinal ﬂuid; ELISA: enzyme-linked immunosorbent assay; RNA: ribonucleic acid electroencephalogram; NS1: nonstructural protein 1; WHO: World Health Organisation. Deﬁnitions. Full recovery: none or slight disability, Partial recovery: moderate or severe disability (may need help walking, numbness, tingling, may need ongoing assistance with daily activities). 3. Results 3. Results [32] 1996 Malaysia Case Report 1 Dengue IgM seropositivity Full recovery Chanthamat et al. [15] 2010 Thailand Case Report 1 NA Full recovery Solomon et al. [33] 2000 Vietnam Prospective Study 1675 IgM/IgG seropositivity (CSF) Partial recovery Sousa et al. [9] 2004 Brazil Retrospective Study 51 Dengue IgM seropositivity (CSF) 49 patients made a full recovery, 2 patients had partial recovery Verma et al. [34] 2011 India Retrospective Study 26 Dengue IgM seropositivity Partial recovery Note: Abbreviations: IV: intravenous; IgM: immunoglobulin M; IgG: immunoglobulin G; NA: not available; CSF: cerebrospinal ﬂuid; ELISA: enzyme-linked immunosorbent assay; RNA: ribonucleic acid electroencephalogram; NS1: nonstructural protein 1; WHO: World Health Organisation. Deﬁnitions. Full recovery: none or slight disability, Partial recovery: moderate or severe disability (may need help walking, numbness, tingling, may need ongoing assistance with daily activities). Table 1. Description of the study design and the reported outcomes. 5 of 11 Pharmacy 2019, 7, 3 Table 2. Characteristics of published studies included in the systematic review. Study Age Sex Number of TM Cases Serotype Treatment Singh et al. [17] 45 Male 1 - T9-11 laminectomy Evacuation of epidural haematoma. Multiple blood and platelet transfusions. Conservative management applied. Ghosh et al. [18] 4 1 - High dose methylprednisolone. Platelet transfusion and Packed red cell Supportive therapy for hepatitis and glomerulonephritis Seet et al. [7] 44 Female 1 - IV methylprednisolone 1 g for 5 days, Spinal MRI, Catheterisation for urinary retention, Intensive physiotherapy Kunishige et al. [13] 42 Male 1 1 IV methylprednisolone Antibiotics Fong CY et al. [19] 12 Female 1 - IV methylprednisolone 30 mg/kg/day for 3 days followed by oral prednisolone IV Immunoglobulin (IVIG) 1 g/kg/day for 2 days. Intubated, 6 cycles of plasma exchange. Cervical epidural haematoma was managed conservatively Gupta et al. [20] 26 Female 1 - Methylprednisolone 1.0 mg/5 days Mechanical ventilation for 2 weeks Wasay et al. [21] 18–35 5 females, 1 male 1 - MRI/CT +/- EEG observations Samanta et al. [22] Male 1 primary/ secondary infection Pulsed methylprednisolone Conservative therapy Misra et al. [23] 5–70. 26 females, 90 males 1 1, 2 and 3 - Sahu et al. [24] 25 +/- 18.3 7 - Symptomatic treatment Soars et al. [25] 11–79. 10 female, 3 male 2 1, 2 and 3 Corticosteroids IVIG Weeratunga et al. [26] Mean: 35 1 female, 6 male 2 - Methylprednisolone pulsed 1 g/3 days Puccioni-Sohler et al. 3. Results 3. Results (Brazil) [12] 22–74 6 females, 4 males 3 - Methylprednisolone 1.0 mg/5 days. Additional Human IVIG 400 mg/kg/5 days for 1 patient Larik et al. [10] 43 Male 1 - IVIG Lim et al. [27] 43 Male 1 - IVIG Tomar et al. [28] 42 Male 1 - IV Methylprednisolone Table 2. Characteristics of published studies included in the systematic review. 6 of 11 Pharmacy 2019, 7, 3 Table 2. Cont. Table 2. Cont. Study Age Sex Number of TM Cases Serotype Treatment Mo et al. [29] 65 Male 1 - IV Methylprednisolone IVIG Plasma Exchange Mota et al. [30] 21 Male 1 - IV Methylprednisolone Leão et al. [14] 58 Male 1 - Ceftriaxone Miranda de Sousa A et al. [31] 11 Female 1 - IV Methylprednisolone 1g/day followed by prednisolone Renganathan et al. [32] 14 Female 1 - Symptomatic treatment Chanthamat et al. [15] 61 Female 1 - IV Methylprednisolone Solomon et al. [33] 2 - Symptomatic treatment Sousa et al. [9] Mean:34 26 3 IV methylprednisolone for 5 days Verma et al. [34] 8 females, 18 males 1 - IV Methylprednisolone Note: Abbreviations: IV: intravenous; IVIG: intravenous immunoglobulin; TM: transverse myelitis; MRI: magnetic resonance imaging; CT: computed tomography; EEG: electroencephalogram; T9–11: thoracic vertebra 9 to 11; kg: kilogram. Pharmacy 2019, 7, 3 7 of 11 A total 2672 cases of Dengue fever in all ages involving 289 (10.8%; (289/2672)) with neurological complications in 25 studies were included in the ﬁnal analysis (Table 2). A total 2672 cases of Dengue fever in all ages involving 289 (10.8%; (289/2672)) with neurological complications in 25 studies were included in the ﬁnal analysis (Table 2). Overall 2.3% (61/2672) had TM, and children (<18 years old) constituted 13% (8/61) of TM cases reported by six studies. Twenty-two studies reported epidemiological data; the neurological complication was twice in males compared to female 67.7% (130/192) vs. 32.7% (62/192) and 1.5-fold increase TM for males 59.3% (32/54) vs. 40.7% (22/54). The mean age at presentation was 33.1years (range: 0.75–61). Of the 19 papers reporting the onset of DF to the time it was complicated by TM, the average was 11.7days (range: 5–42). All the studies reported method of diagnosing TM, and apart from the use of radiological investigation by all the studies, the method of diagnosing TM due to DF was mainly IgM seropositivity (92% (n = 23/25)). 3. Results 3. Results In addition, 12 papers mentioned additional methods were also used in diagnosing cases of TM; cerebrospinal ﬂuid analysis (CSF) analysis (nine studies), IgG antibodies (two studies), clinical features and nonstructural protein 1 (NS1) antigen assay (two studies) with one other study which used an antibody index ratio of IgM to IgG. y g g Out of the 25 studies, 92% (n = 23/25) speciﬁed their management plans. High dose methylprednisolone was used in 82.6% (n = 19/23) of studies with additional antibiotic cover. Twenty-two per cent (n = 5/23) of studies required in addition intravenous immunoglobulins, of which two had assisted ventilation and one had blood/platelet transfusions. Three studies employed a symptomatic management plan. Only one study treated with antibiotics only and a laminectomy was a modality of management in one of the studies. The commonest treatment modality was steroids 82.6% (n = 19/23). In terms of recovery after, only half had full recovery 50.8% (n = 31/61) from TM. There was no mortality following dengue infection reported, however, the crude case fatality rate following TM was 3.3% (n = 2/61), involving a 45-year-old male and a 9-month-old male infant. 4. Discussion This supports the earlier studies indicating that other factors including biology, environment and 8 of 11 Pharmacy 2019, 7, 3 experience are contributors to human health [40], but contrary to the reports that most autoimmune diseases are more frequent in females than in males [41]. There is currently no agreed consensus on the management of TM. Our ﬁndings showed that almost 80% cases were treated with high dose of methylprednisolone despite insufﬁcient evidence regarding the utility of steroids in treating transverse myelitis [42]. It is therefore advisable that until more robust evidence is available, administration of high dose intravenous (IV) methylprednisolone will be the ﬁrst treatment of choice in TM to enhance neurological functions. Few cases however required immunoglobulins but this was introduced at a later stage, and to those cases that are presumed to be very sick, thus assessing the efﬁcacy at this stage becomes difﬁcult. This has been considered mainly as second line therapy in patients who have not recovered or are poorly recovering from TM [42]. Due to the supposedly rarity of TM associated DF, there has been controversy as to the actual prevalence of TM following DF. de Sousa AM [9] and colleagues in a retrospective study conducted in the Brazilian Amazon region showed almost half of all DF cases had TM following DF (44%, 26/59). This was adduced to an epidemic of DF at the time of the study compared to the study in a tertiary centre in India where of the 116 patients with DF only 1% had TM. Our review of 2.3% of TM-associated DF may have been underestimated and should therefore be interpreted with caution, since some post infectious TM have been known to present even months after the primary DF infection [23,31]. More importantly, is the signiﬁcant morbidity associated with TM following DF, as only half had a full recovery from TM before discharge with 19.7% with no reported recovery. This highlights the need for a careful evaluation of patients with DF for TM and other possible neurological complications, and prompt management as high dose steroids has shown to be effective, especially if instituted early in the management of suspected cases of TM following Dengue. However, our results demonstrate the strengths of combining outcomes of rare events through a detailed systematic review of the literature. 4. Discussion A detailed systematic review of the literature identiﬁed all reported cases of TM following DF in endemic countries irrespective of the mode of presentation. Overall there were 61 cases in the literature, accounting for 2.3% of DF and the crude case-fatality rate among TM cases was very low at 3.3%. These ﬁndings, contrary to previously reported rare occurrence, conﬁrm the prevalence of TM following Dengue. Moreover, Dengue is the most common arboviral disease [35], and occurs in Southeast Asia, East and West Africa, the Caribbean and the Americas [36]. Interestingly, the majority of TM cases were in Asia and a few reported cases in North America. One of the explanations for the low prevalence of TM following DF in West Africa and the Caribbean is that the Asian population appears to be prone to autoimmune injury of the spinal cord and some genetic make-up, including the type of Dengue that causes TM might be different and their contributing factors. Although, neurological complications in dengue fever have been documented with all serotypes, we also observed that it is more common with serotypes 2 and 3 [33]. yp For example, of the four strains of dengue virus implicated in the disease, DEN3, DEN2 and DEN1 are the prominent serotypes in India. The DEN2 has been reported in more than 75% of the cases in breakouts since 2010 [37,38]. A similar ﬁnding was observed in the review with serotype 2 having been the most commonly isolated, however, this was only reported in ﬁve of the studies. The mechanisms of viral transmission and spinal cord injury induced by dengue virus are unclear. Two mechanisms have been postulated: by direct invasion of the cord and by active replication within the spinal cord [13], which is common during the early phase or postinfectious immune injury [39]. Since only ﬁve studies were able to isolate dengue IgG/IgM or antigen in the CSF, it is therefore most likely that both mechanisms have been implicated in the cases in this review. One important ﬁnding is the 2-fold increase in neurological complication, and a 1.5-fold increase in those that had TM in females compared to males. In addition, the two mortalities were in males. 4. Discussion The large number of case reports and lack of observational studies was a limitation; consequently, we were unable to conduct any meta-analyses to compare differences in other TM-associated neurological complications or calculate risks associated with clinical outcomes. In addition, as would be expected from case reports, several of the population denominators were not available to identify cases; this could potentially lead to double counting of the same cases. Therefore, it is important that future studies report the number of cases of DF during the time period so that TM rate can be calculated and compared in different population. 5. Conclusions This review highlights the risk of TM following dengue. Although neurological complications are rare, especially TM, once set in, they are associated with signiﬁcant morbidity. A high index of suspicion is therefore required with careful evaluation and follow-up of patients, as well as, prompt management to enhance recovery. Author Contributions: N.B. reviewed the literature, analysed the data, was involved in the interpretation of the data, writing the report (including the ﬁrst draft), coordinated the production of the manuscript, had full access to all the data in the study, takes responsibility for the integrity and accuracy of the data analysis and approved the ﬁnal manuscript as submitted. D.A. and O.O. carried out the initial analyses, were involved in the interpretation of the data and writing of the report and approved the ﬁnal manuscript as submitted. G.O. conceptualised and designed the study, was involved in the interpretation of the data and writing the report, coordinated the production of the manuscript, had full access to all the data in the study, takes responsibility for the integrity and the accuracy of the data analysis and approved the ﬁnal manuscript as submitted. All authors approved the ﬁnal manuscript submitted and also agreed to be responsible for all aspects of the work. Funding: No external funding was received for the design, conduct of the study, collection, management, analysis, and interpretation of the data; preparation, review or approval of the manuscript; and decision to submit the manuscript for publication. Conﬂicts of Interest: The authors declare no conﬂicts of interest. 9 of 11 Pharmacy 2019, 7, 3 Pharmacy 2019, 7, 3 Appendix A Appendix A pp Table A1. FreeText and MeSH search terms. Category Search Terms Transverse Myelitis “transverse myelitis” or “myelitis” or “TM” or “Myelitis, Transverse/” Dengue Virus “DEN” or “DHF” or “Severe Dengue/” or “Dengue Virus/ Table A2. FreeText and MeSH search terms results. Search Results Transverse Myelitis Category 146,970 Dengue Virus Category 83,794 Transverse Myelitis AND Dengue Virus Category (ENG) 226 (Additional publications from contacts) 16 Duplicates Removal (62) 180 Limits Applied and Studies Removed (136) 44 Title and Abstract Exclusion (18) 26 Full Text Exclusion (1) 25 Table A1. FreeText and MeSH search terms. References 1. Bhatt, S.; Gething, P.W.; Brady, O.J.; Messina, J.P.; Farlow, A.W.; Moyes, C.L.; Drake, J.M.; Brownstein, J.S.; Hoen, A.G.; Sankoh, O.; et al. The global distribution and burden of dengue. Nature 2013, 496, 504–507. 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https://openalex.org/W3167560796	http://dbc.wroc.pl/Content/110254/Mugosa_Popovic_Deviation_from_target_capital_structure_as_a_factor.pdf	English	null	Deviation from target capital structure as a factor of acquisition decisions in European developed markets	Argumenta Oeconomica	2,021	cc-by-sa	10,476	ARGUMENTA OECONOMICA No 1 (46) 2021 PL ISSN 1233-5835 ARGUMENTA OECONOMICA No 1 (46) 2021 PL ISSN 1233-5835 ARGUMENTA OECONOMICA No 1 (46) 2021 PL ISSN 1233-5835 Ana Mugoša, Saša Popović DEVIATION FROM TARGET CAPITAL STRUCTURE AS A FACTOR OF ACQUISITION DECISIONS IN EUROPEAN DEVELOPED MARKETS The purpose of this paper is to examine the causal relation between deviations from target capital structure (leverage deficit) and acquisition choices in capital markets in Western Europe. The analysis is conducted using a sample of 921 large companies, which represents a strong and solid base for testing target capital structure and takeover interdependence, as the focus is on the period when half of the largest M&A deals in Western Europe occurred. This study found that leverage deficit is a crucial determinant of acquisition choices and market reaction on acquisition announcements, measured by CARs to bidders. Companies that are underleveraged relative to their target capital structure have a higher probability of undertaking acquisitions. On the other hand, the market reacts unfavourably to acquisition announcements of underleveraged acquirers – overleveraged companies undertake the most value-enhancing acquisitions, whilst underleveraged companies make poor acquisition choices. This paper enriches the literature by empirically extending the understanding of how managers make investment decisions in relation to capital structure, and how capital markets assess the impact of these investments. Keywords: capital structure, acquisitions, leverage deficit, Western Europe, capital marke cumulative abnormal returns.i JEL Classification: G10, G14, G15, G30, G32, G34 DOI: 10.15611/aoe.2021.1.03 JEL Classification: G10, G14, G15, G30, G32, G34 DOI: 10.15611/aoe.2021.1.03 JEL Classification: G10, G14, G15, G30, G32, G34 DOI: 10.15611/aoe.2021.1.03 * Faculty of Economics, Department of Finance, University of Montenegro, Montenegro. 1. INTRODUCTION This paper aims to fill the gap in the analysis of shaping capital structure in relation to M&A, by enriching the existing research with the leverage deficit concept, and estimating the effects of management investment decisions in European capital markets. Corporate restructuring through acquisitions represents the primary way in which companies may increase business value and provide returns to their shareholders. Being the largest investment decisions that companies can make, acquisitions are very complex transactions which require both decision- 54 A. MUGOŠA, S. POPOVIĆ making on strategy choice and sources of finance. Numerous studies (Andrade et al., 2001; Eckbo, 2009; Martynova and Renneboog, 2009; Hu and Yang, 2016) have highlighted the strong relation between capital structure (sources of finance) and corporate takeovers. Even though these topics have been examined over the years, there are only a few academic papers that analysed target capital structure as a factor of the acquisition decision-making process (Maloney et al., 1993; Harford et al., 1999; Uysal, 2007, 2011; Harford, 2009). It is important to point out that Uysal (2007, 2011) gave the first results on the effect of leverage deficit (the deviation from target debt or leverage) on acquisition decisions and no similar research has been conducted after 2011 (to the best of this author’s knowledge). The paper represents one of the most cited and influential articles in the field of M&A and target capital structure interdependence. To sum up, there is an evident lack of such research in developed countries of Western Europe, with only a few research papers analysing the relation between target capital structure and acquisition choices, and only one which gives the results on the actual impact of leverage deficit on acquisition decisions. The optimal choice of the financial resources mix has become one of the most crucial scientific dilemmas, overcoming its basic function of debt and equity choice. The issue of optimal capital structure has been the focus of numerous financial studies, yet it still remains unresolved. Considering these facts, this paper aims to shed light on the causal relation between the leverage deficit of the bidder and the resulting acquisition choices made accordingly, as well as to test the market reaction, measured by changes in the bidders’ stock price in Western Europe. The analysis focuses on the period of 2003-2010, which incorporates the sixth M&A cycle. 1 Bloomberg database and Institute for Mergers, Acquisitions and Alliances, IMAA; https:// imaa-institute.org/mergers-and-acquisitions-statistics/ 1. INTRODUCTION Even though it is important to have an up-to-date sample, the relevant research regarding M&A consists of the periods with important changes in M&A transactions – seven of the ten largest M&A deals worldwide were completed from 1998 to 2007, and five of the ten largest M&A deals in Western Europe were completed in 2003-20101. Consequently, this sample represents a strong and solid base for the estimation of target capital structure. Additionally, it is also extremely important to analyse previous periods, in order to understand the causes of the current results in the M&A segment. This paper tests two research questions/hypotheses:ii 1. Is leverage deficit a significant factor in the process of acquisition choices (are underleveraged companies more acquisitive)? 55 DEVIATION FROM TARGET CAPITAL STRUCTURE... 2. How do capital markets react to the acquisition announcements of underleveraged companies? 2. How do capital markets react to the acquisition announcements of underleveraged companies? The research is based on prior analysis conducted by Uysal (2007, 2011). The first part of the analysis tested the probability of acquisition depending on the acquirers’ leverage deficit. In the second part, the market reaction to the acquirers’ announcements was tested, calculating the cumulative abnormal returns to bidders over a five-day event window. The sample consists of 921 large bidders, which gives the possibility to precisely test debt ratios, as large companies are prone to significant changes in capital structure, especially during corporate takeovers. Compared to previous research (Mugoša, 2015), this study used the estimation of capital structure determinants as a crucial factor to determine leverage deficit and to generate a completely new and more complex methodology and analysis –to test the probability of making an acquisition depending on the deviations from target debt ratio, and the capital market’s reactions to these announcements. The contribution of this paper is in the estimation of the target capital structure and investment decisions’ interdependence in the developed European markets, which has not been researched as much as North American market, specifically in the context of fluctuations in actual debt ratio (leverage deficit). Furthermore, mergers and acquisitions are a vital part of today’s global economy across various industries. This is the reason why these transactions are very interesting to focus on in research. Additionally, detailed and comprehensive data generated from acquisitions createsthe opportunity to conduct objective and in-depth analysis. 2. THEORETICAL BACKGROUND Starting from Miller and Modigliani (1958) and the irrelevance proposition, capital structure research has been continuously conducted over five decades, leading to the formulation of contemporaneous models and theories. The central theme analysed in the literature is the possibility of capital structure optimization. A comprehensive model which incorporates all empirical observations does not exist, therefore each company must develop financial flexibility and set targets under imposed specific conditions (Myers, 1984).l Empirical research suggests that the real situation on the market is reflected in the mix of different theories (Graham and Harvey, 2001; Bancel and Mittoo, 2004; Brounen et al., 2006). Testing the application of capital structure theoretical concepts showed that companies, especially large ones, define target debt ratio, ranging from strict or somewhat strict to flexible. On the other hand, the main factors affecting capital structure decisions are financial flexibility (preserving unused debt capacity) and credit rating. This means that depending on financial flexibility, transactions, agency and financial distress costs, companies tend to adjust their target capital structure (Kayhan and Titman, 2007). The results lead to the conclusion that empirical evidence is not in favour of any single theory. Still, it is important to notice that the finance theory is more applicable regarding larger companies and that the majority of companies have target debt ratios. p g Various studies (Hovakimian et al., 2001; Fama and French, 2002; Leary and Roberts, 2005; Kayhan and Titman, 2007; Uysal, 2007, 2011; Harford et al., 2009; Hernádi and Ormos, 2012; Tao et al., 2017) found that companies adjust capital structure and move towards target debt ratios, specifically due to different corporate activities. Bearing in mind that capital structure is an important factor in determining if a company becomes a target or bidder, adjusting capital structure can be used as a financial motive prior to, and post M&A transaction. The findings from Yang (2011) suggest that companies approach their target leverage ratios when acquiring other firms. Vermaelen and Xu (2014) state that acquirers in 80% of cases move toward target debt ratios and that the payment made in cash, which is mostly used by underleveraged bidders, is common compared to equity financing. Bidders have significantly lower leverage ratios, while in the meantime a larger financial slack, prior to acquisitions. Accordingly, cash-rich bidders are more likely to acquire a slack-poor target, due to the greater access and availability of financial sources (unused debt capacity). 1. INTRODUCTION The empirical verification of the model could be of use for managers in the process of evidence-based capital structure decision-making, by facilitating the decision as to what are the most important factors of optimal capital structure, and how this structure can accordingly impact on future investment decisions. Finally, the results of this research could be used in order to make a comparison with the US research (to test the differences between these two markets), taking into account the global investments of multinational companies, as well as to call for this analysis in Central and Eastern Europe. The reminder of the paper is organized as follows: Section 2 presents the relevant theoretical and empirical evidence concerning capital structure and corporate restructuring. Section 3 describes the used methodology (FGLS – target debt estimation, probability analysis, event study). Section 4 generates the results and discussion. Section 5 provides the final conclusion and direction for future research. 56 A. MUGOŠA, S. POPOVIĆ 2. THEORETICAL BACKGROUND Leary and Roberts (2005) also confirm adjustments toward an optimal range of capital structure, due to possible profitable investments, whereas it is important to increase financial 57 DEVIATION FROM TARGET CAPITAL STRUCTURE... flexibility. Higher financial flexibility definitely goes along with lower leverage, which is essential to acquirers when deciding to enter in the process of M&A (Bouraoui and Li, 2014).i Uysal (2007, 2011) specifically emphasizes the importance of the link between the deviations from target capital structure or leverage deficit and corporate investments. The current capital structure may impact on future investment opportunities or M&A, especially when financial flexibility varies depending on present market frictions (Baker and Martin, 2011). Constrained financial flexibility leads to a deviation from target debt ratio, which consequently may determine the possibility of the company to engage in M&A activities, as well as define its position in these transactions – being a bidder or a target. This is why a deviation from the target capital structure plays an important role in corporate investment decisions. For example, an overleveraged company will probably be unable to perform a takeover, being a cash-poor firm, and in this situation it is forced to use equity financing, which is the costliest source of financing. These companies are less likely to engage in M&A activities, compared to underleveraged or cash-rich firms. Accordingly, the results from Uysal’s (2007) research suggest that underleveraged companies are more acquisitive (one standard deviation decrease in leverage deficit increases the likelihood of making an acquisition by 7.4%). Underleveraged companies have higher financial flexibility and more financing sources available. The author also concluded that underleveraged bidders acquire larger targets and engage in the process of acquisition more frequently. However, a challenging and very important question in this context is, what are the valuation effects on capital markets when considering the capital structure’s impact on M&A transactions (bidder’s performance)? M&A’s basic goal is to produce positive effects and increase the value of a new company through synergy. Due to market imperfections, these transactions will be assessed according to the chosen financial mix, with respect to the agency costs. It must be taken into account that stock prices play an important role in determining capital structure, especially if the company considers issuing equity. This is why changes in stock prices must be considered in order to test the success of transaction. 2. THEORETICAL BACKGROUND M&A valuations are mostly estimated using an event study, measuring the cumulative abnormal returns of acquirers around the event of takeover. Uysal (2007, 2011) indicated that stock prices decrease in cases of acquisition announcements of underleveraged companies. This result is consistent with Jensen’s (1986) free cash flow hypothesis, which states that cash-rich firms may make negative investment choices that benefit them personally, namely the increase of free cash flow agency costs. Various 58 A. MUGOŠA, S. POPOVIĆ studies focused on the impact of leverage on the returns of both the bidder and the target. Maloney, McCormick and Mitchell (1993) reported higher acquisition returns with the greater increase in bidders’ leverage. Smith and Kim (1994) found that the average returns of bidders with high free cash flows are negative, which leads to inefficient NPV choices (paying too much for a target). This means that slack-poor bidders (higher leverage) have higher positive total returns. As the authors stated, restraining management by decreasing liquidity and increasing leverage in companies with high free cash flows, will increase the shares’ value. Consistent with the arguments of Jensen (1986), Kang (1993) reported that the returns of Japanese bidders when they acquire US targets, increase with the bidders’ total debt (additionally, the ties to financial institutions and the depreciation of the dollar had a positive effect on the bidder’s returns). The research most closely related to Uysal’s paper, conducted by Harford (1999), also resulted in a negative market reaction to acquisition announcements of cash-rich firms, whereas cash-richness was a strong predictor of the acquisition likelihood. The evidence from this paper was consistent with the explanatory power of the free cash flow hypothesis. Yet Harford did not apply deviations from target capital structure as a factor, but focused on the deviation of a firm’s cash reserves from its predicted optimal level. Harford, Klasa and Walcott (2009) implemented deviations from the target capital structure, and their evidence is in line with the previously considered abnormal returns, as well as with the trade-off theory, as bidders rebalance capital structure towards the target after the takeover. The more recent paper by Beschwitz (2018) concluded that cash windfalls cause the decrease of abnormal returns to bidders (overinvestment). These results are consistent with the view that high leverage can be used as a disciplinary mechanism that will decrease agency costs and problems between managers and shareholders. 2. THEORETICAL BACKGROUND The general conclusion is that leverage plays an important role in the process of acquisition choices and outcomes. Lower leverage means the higher probability of acquisitions, while increased leverage is associated with higher abnormal returns, indicating that managers have made investments which increase shareholders’ value (Durand et al., 2016; Uysal, 2007, 2011; Ahmed and Elshandidy, 2018). Finally, the leverage deficit can be used as an efficient and significant determinant, because it subsumes the effects of both excess cash reserves and leverage. The importance of leverage as a factor of investment decisions was also confirmed in the paper by Harford and Uysal (2014), which broadened the analysis to the impact of debt market access on the ability to make investments. The results suggest that constrained access to debt leads to underinvestment. 59 DEVIATION FROM TARGET CAPITAL STRUCTURE... The findings of this paper aim to solve the research gap found in the relation between the deviations from the target capital structure and M&A in Western European capital markets. 2.1. Determinants of target debt ratio POPOVIĆ implying a negative relation between stock returns and debt (Hovakimian et al., 2001; Baker and Wurgler, 2002; Welch, 2004; Uysal, 2007). Stock return is defined as the average adjusted percentage annual stock return. Size represents another important factor of capital structure. Large companies have less volatile cash flows, lower bankruptcy and financial distress costs, and easier access to sources of financing (Rajan and Zingales, 1995; Uysal, 2007, 2011; Frank and Goyal, 2009; Kirch et al., 2012). Easier access to creditors is gained through the more transparent and diversified business of these companies, compared to small ones, which decreases information asymmetry and agency costs (large companies’ shares are often traded in the stock market). This implies a positive correlation between size and target leverage. The impact of size determinant is proxied by the natural logarithm of sales.i Profitability is assumed to be positively correlated to debt, as more profitable firms are less prone to bankruptcy (Frank and Goyal, 2009). On the other hand, firms prefer internal over external funds (according to the pecking- order theory) and this fact suggests a negative relation between profitability and target debt ratio (Kędzior, 2012). Profitability will be proxied by earnings before taxes, preferred dividends and interest payments over total assets. i Product uniqueness is a measure of the unique and specific business (production, distribution). Unique products require higher costs of production and trade, leading potentially to higher costs of bankruptcy and financial distress (Titman and Wessels, 1998). Consequently, access to creditors is limited, decreasing target leverage ratio. Thus it is assumed that uniqueness and leverage are negatively correlated. The proxy for this determinant will be the ratio of research and development (R&D) expenditure to total assets. 2.1. Determinants of target debt ratio In order to estimate the effects of leverage deficit on acquisition choices, target debt ratio is regressed on a set of potential determinants of capital structure, used in previous studies (Rajan and Zingales, 1995; Hovakimian et al., 2001; Uysal, 2007). The determinants will include proxies for tangibility (tangible assets ratio), growth, size, profitability and product uniqueness. The first four determinants are included in the core model of leverage (Frank and Goyal, 2009), a model which consists of the most important capital structure factors. The reasoning behind this model is the fact that these variables/factors explain over 30% of variations in leverage. The results from previous studies show that tangibility, growth, size and profitability are constantly applied in models, whereas other factors vary depending on the specific research (expected inflation, tax rates, product uniqueness, cash flow volatility etc.). Tangibility of assets represents an important capital structure determinant, as companies with a higher share of tangible assets are likely to have higher leverage. Tangible assets as collateral provide better access to creditors, lower bankruptcy and financial distress costs. When the debt issued is secured by the property with known values (liquid assets), these firms are more likely to have lower bankruptcy and agency costs, and hence higher target debt ratios (Titman and Wessels, 1998; Uysal, 2007). On the other hand, if the structure of debt is being considered (short-term debt), the specific industry of the company (illiquid assets), tightened banks’ credit activity and the illiquid capital market, collateral can be negatively related to target debt ratio. In this study, the ratio of tangible assets to the book value of total assets will be used as a proxy for tangibility. Company’s growth affects capital structure. High level of leverage could potentially limit growth and prevent future profitable investment. The costs associated with the agency conflict are particularly higher for growing firms and industries (Titman and Wessels, 1998; Uysal, 2007; Kirch et al., 2012). Accordingly, it can be assumed that growth opportunities will be negatively related to target debt. ‘Market to Book’ (ratio of company’s market value to its total assets) and ‘Total Return’ (average stock price return) were used as proxies for growth (Uysal, 2007). Changes in stock prices can significantly impact capital structure as they serve as a proxy of the company’s market performance. Sudden increases in stock prices (stock run-ups) cause shares issuance, 60 A. MUGOŠA, S. 3.1. Sample selection The estimation of target leverage ratio was conducted through the analysis of the annual panel data of companies in Western Europe covering the period from 2003 to 2010. As mentioned in the introduction, this sample represents a solid base for the analysis of target capital structure, as the majority of the largest M&A deals in Western Europe were completed in that period. The data was generated from Bloomberg Annual Files. Companies which met the following criteria were included in the sample: 1. actively trading stocks in observation period; 2. companies domiciled in Western Europe; 61 DEVIATION FROM TARGET CAPITAL STRUCTURE... 3. companies not operating within the financial sector and regulated utilities; 4. types of securities traded are common stocks;i 5. the value of sales in the fiscal year 2010 was greater than or equal to 50 million euros2;i 5. the value of sales in the fiscal year 2010 was greater than or equal to 50 million euros2;i 6. market capitalization in the fiscal year 2010 was greater than or equal to 400 million euros3. 6. market capitalization in the fiscal year 2010 was greater than or equal to 400 million euros3. After meeting these criteria, the sample created consisted of 921 large companies that were engaged in acquisition activities. Large companies provide the possibility to precisely test debt ratios, as they are prone to significant changes in capital structure especially during corporate restructuring. Target leverage ratio was estimated by testing following fundamental capital structure indicators/determinants: 1. Book leverage (BL) defined as Total Liabilities over Total Assets.i 2. Tangibility (TA) defined as Tangible Assets over Total Assets.i 3. Market to Book (MB) defined as Total Market Value over Total Assets. 4. Sales are the natural logarithm of Sales, Revenue or Turnover.ii 5. Profitability defined as EBITDA over Total Assets.i 6. Product Uniqueness (PU) defined as R & D Expenditures over Total Assets. 7. Total Return /average stock returns (TR). 7. Total Return /average stock returns (TR). 2 Based on the rules adopted by the European Commission in 2005, still in force, a company is defined as small and medium sized (SME) if its annual income from sales is less than or equal to 50 million euros. 3 Even though there is no consensus on a lower and upper market capitalization limit, this value for SMEs in Western Europe ranged, on average, from 100 to 200 million euros, while in 2010 it amounted to 400 million euros on average. These companies were excluded from the sample for two reasons: (1) to focus on financially stable companies, which are most probably large companies; (2) a negligible proportion of small companies would, through extreme values in the model, create the effect of noise (noisy variables). to 50 million euros. 3 Even though there is no consensus on a lower and upper market capitalization limit, this value for SMEs in Western Europe ranged, on average, from 100 to 200 million euros, while in 2010 it amounted to 400 million euros on average. These companies were excluded from the sample for two reasons: (1) to focus on financially stable companies, which are most probably large companies; (2) a negligible proportion of small companies would, through extreme values in the model, create the effect of noise (noisy variables). 2 Based on the rules adopted by the European Commission in 2005, still in force, a company is defined as small and medium sized (SME) if its annual income from sales is less than or equal to 50 million euros. 3 Even though there is no consensus on a lower and upper market capitalization limit, this value f SME i W t E d f 100 t 200 illi hil i 2010 3.2. Target debt ratio estimation procedure Previously conducted empirical research suggests that companies have targets related to debt ratio. US evidence shows (Graham and Harvey, 2001) that the target exists as strict (10%), somewhat tight (37%) and flexible (37%). In Europe, approximately 75% of companies have somewhat tight to flexible target debt ratios (Bancel and Mittoo, 2004; Brounen et al., 2006). Accordingly, the adjustments and deviations from target debt ratios can be explained by various theoretical reasons: information asymmetry, market timing, free cash flow etc. Consistent with empirical research and capital structure theoretical concepts, 62 A. MUGOŠA, S. POPOVIĆ in the first phase the target or optimal leverage ratio is estimated by regression of book leverage of company i on a vector of explanatory variables: 1 2 3 4 5 6 . & i i i i i i i i i i i Tangible Assets Book leverage Marketto Book LogSales Total Assets Ebitda R D AverageStock Return Total Assets Total Assets = + + + + + + + +  α β β β β β β ε (1) (1) The model includes book leverage as a dependent variable. First, there is a mechanical relationship between market leverage and the determinants expressed in market values (e.g. market to book). If market value increases, spurious correlation can occur between the variables scaled by market value, increasing both the left and the right side of regression (Parsons and Titman, 2009). Additionally, due to steep run-ups of stock prices, market leverage regression results can lead to confusing results, meaning that some firms could be potentially identified as underleveraged. Finally, studies show that the book value of leverage proved to be suitable in testing assumptions of capital structure theories (Fama and French, 2002). In order to capture and control the effects of the time-invariant characteristics of unobservable firm-specific factors, the model was primarily estimated with the Fixed-Effects Panel Method (FE). The time-invariant characteristics of unobservable firm-specific factors explain most of the cross-sectional variations in capital structure and the potential deviation from the target capital structure. These characteristics are the company’s sector of activity, managerial skills, managerial abilities etc. 3.2. Target debt ratio estimation procedure Recent empirical evidence (Flannery and Rangan, 2006; Lemmon et al., 2008; Chang and Dasgupta, 2011), shed some light on the reliability of OLS and the Tobit regression and the explanatory power of capital structure determinants, as these methods estimates may be biased because of their not controlling for omitted variables (unobservable firm-specific factors). However, the results of FE were subject to bias, because FE post-estimation indicated the presence of heteroskedasticity and first-order autocorrelation. This was the reason to apply the Feasible Generalized Least Squares Method (FGLS). FGLS allows estimation in the presence of AR(1) autocorrelation within panels and cross-sectional correlation and heteroskedasticity across panels. Being based on the calculation of panel- specific residual variance structure, FGLS could potentially absorb the fixed effects for the panel variable specified. FGLS is defined as (Baltagi, 2013): ( ) 1 FGLS X X X y β − ′ ′ = Ω Ω, ( ) 1 FGLS X X X y β − ′ ′ = Ω Ω, (2) (2) where: X represents independent variable, Y dependent variable and Ω covariance matrix of unique errors. where: X represents independent variable, Y dependent variable and Ω covariance matrix of unique errors. 63 DEVIATION FROM TARGET CAPITAL STRUCTURE... 3.3. Univariate analysis and multivariate analysis – probit This stage of the analysis gave the answer to the first research hypothesis: is capital structure a significant factor in the acquisition process, and if confirmed, are underleveraged companies more acquisitive? The probability of making an acquisition was tested by univariate and multivariate (probit) analysis. Univariate analysis is a simple form of statistical analysis, which describes the frequency and distribution of the data. Multivariate models or binary choice models are applied in cases of a binary dependent variable – a variable taking the value 0 or 1. The probability of the dependent variable taking one of two values is estimated using the probit or logit model (Wooldridge, 2012). ( ) ( ) β x β x lim Prob γ 1 1 and lim Prob γ 1 0 ∞ ∞ →+ →− ′ ′ = = = = . (3) (3) The probit model is defined as: The probit model is defined as: ( ) ( ) ( ) ( ) β x β x 2 1 Prob Y 1 exp t / 2 dt t dt β x 2π ∞ ∞ φ φ ′ − − ′   = = − = =    ′ ∫ ∫ (4) (4) where: φ(t) is standard normal density, and φ is standard normal cumulative distribution. Cumulative distribution function for a discrete variable is given by: ( ) ( ) ( ) F a P x a , whereas 0 F a 1 = ≤ ≤ ≤ . (5) (5) The function is non-decreasing and probability distribution is computed as: The function is non-decreasing and probability distribution is computed as: ( ) i i x a F a p ≤ = ∑ . (6) (6) 3.4. Market valuation of M&A – event study Event studies are used to assess the impact of an event (M&A, stock split, earnings and dividends announcements) on the value of a company and its stock price. The aim of an event study is to determine whether, due to a certain event, there exists an abnormal stock price performance or abnormal return, in the analysed period of time, i.e. an event window. If the market is efficient, the effects of the event will be immediately reflected in stock price changes. In this manner, in a relatively short period it is possible to observe the significant economic impact of the event which occurred. The event study was first applied by Fama et al. (1969), to test the market efficiency or the adjustment speed of stock prices to stock splits announcements. The authors 64 A. MUGOŠA, S. POPOVIĆ used the market model to assess abnormal returns, now one of the most common models used. In the first step the expected return on stock i in month t, E(Rit) is calculated using a benchmark, stock market index Rmt: ( ) it i mt it E R R u α β = + + (7) (7) In the second step, the abnormal return ARit is derived as the difference between the actual stock return Rit and the expected stock return E(Rit): In the second step, the abnormal return ARit is derived as the difference between the actual stock return Rit and the expected stock return E(Rit): ( ) it it it AR R E R = − (8) (8) Accordingly, one generates average abnormal returns ARt for each company i in period t (N represents the number of companies): 1 1 N t it i AR Ar N = = ∑ (9) (9) Finally, in the third stage the average abnormal returns are added up in order to obtain cumulative abnormal returns, or the information cumulative effect on the stock prices. The information effect is measured in period t1-t2 (the days before and after announcement day t0): 2 1 2 1 , t t t t t t CAR AR = =∑ (10) (10) The final step is the test of abnormal and cumulative abnormal returns of statistical significance, using the ordinary least square method. In this context the results of the event study give the answer to the second research hypothesis related to the market reaction on the acquisitions announcements. DEVIATION FROM TARGET CAPITAL STRUCTURE... Table 1 Summary statistics of 921 companies from 2003-2010 Value BL TA MB Log Sales Profitability PU TR Number of companies 921 921 921 921 921 921 921 Number of company’s yearly observations 7048 7048 7048 7034 7048 7048 6508 Average 0.599 0.755 1.378 9.229 0.145 0.020 26.526 Median 0.604 0.832 1.072 9.192 0.128 0.000 21.404 Standard deviation 0.205 0.251 1.635 0.687 0.137 0.057 61.771 Variance 0.042 0.063 2.674 0.472 0.019 0.003 3815.652 Coefficient of variation 0.342 0.332 1.187 0.074 0.945 2.856 2.329 Q1 0.485 0.650 0.748 8.776 0.092 0.000 -4.724 Q3 0.713 0.944 1.603 9.698 0.179 0.020 49.348 Interquartile difference 0.228 0.295 0.855 0.922 0.087 0.020 54.072 Source: author’s calculations. companies being underleveraged or overleveraged. The mean values of explanatory variables TA, MB, Log Sales, Profitability, PU and TR are 0.755, 1.378, 9.229, 0.145, 0.020 and 26.526, respectively. 4.1. Descriptive statistics The first results are given through the summary statistics of companies from the sample as shown in Table 1. The book leverage on average amounts to 0.599 with a high standard deviation of 0.205. The large variance around the mean could imply that a group of companies potentially deviate from the target debt ratio. Potentially, one could presume that, according to these changes, the sample consists of DEVIATION FROM TARGET CAPITAL STRUCTURE... 65 4.2. Target leverage ratio estimation In the first phase, FGLS is applied in the target leverage ratio estimation procedure. The FGLS estimator could inflate the chi-square statistic if the dummy variables for firm-specific factors are included. Therefore, two models were estimated: Model 1 without dummy variables and Model 2 including industry and time dummies (controlling for industry effect and macroeconomic changes over the years). Table 2 summarizes the results of the coefficient estimates of the yearly target leverage ratio regressions. The results are similar in Model 1 and Model 2: each determinant is statistically significant in terms of sign and correlation to target debt ratio. Even though the inclusion of dummies did not significantly impact on the coefficient results, it did inflate the chi-square statistics (varying from 843.33 in Model 1 to 3596.43 in Model 2). Therefore Model 1 was used as the further basis for leverage deficit’s impact estimation. 66 66 A. MUGOŠA, S. POPOVIĆ Table 2 Regression estimates of the target leverage ratio using FGLS Variables FGLS (1) FGLS (2) Tangibility -0.0143* -0.00910 (0.00330) (0.00362) Market to Book -0.00941* -0.00426* (0.00114) (0.00100) Logarithm of sales 0.0657* 0.0702* (0.00301) (0.00311) Profitability -0.0759* -0.0749* (0.00874) (0.00866) Product uniqueness -0.0875* -0.0593* (0.0226) (0.0191) Total return -6.25e-05* -2.86e-05 (1.02e-05) (1.15e-05) Constant 0.0239 -0.0242 (0.0287) (0.0294) Number of observations 6,482 6,482 Number of variables 879 879 Panels Heteroskedastic Heteroskedastic Correlation Common AR(1) coefficient for all panels (0.7888) Common AR(1) coefficient for all panels (0.7471) Estimated covariances 879 879 Estimated autocorrelations 1 1 Wald χ2 (6) 843.33 3596.43 Prob > χ2 0.0000 0.0000 Note: Standard errors in parentheses. * p<0.01, p<0.05, * p<0.1/ S th ’ l l ti Table 2 Regression estimates of the target leverage ratio using FGLS Table 2 The results of the coefficient estimates are largely consistent with those of previous studies (Hovakimian et al., 2001; Kayhan and Titman, 2007; Uysal, 2007, 2011; Frank and Goyal, 2009; Hernádi and Ormos, 2012; Kirch et al., 2012). Target debt ratio increases with sales (0.0657, p-value less than 0.001). Presumably, larger companies have a higher debt capacity and easy access to sources of finance caused by less volatile cash flows and diversified business (lower information asymmetry). Market to book has a negative relationship with 67 DEVIATION FROM TARGET CAPITAL STRUCTURE... target leverage (-0.00941, p-value less than 0.001). Highly market valued companies prefer internal funds or more probably equity issuance. 4.2. Target leverage ratio estimation Reducing leverage is logical in periods of favourable market conditions (high equity market value). This result is consistent with market timing (Baker and Wurgler, 2002). Along with market to book, total return (average annual stock return) is a proxy for growth and is negatively correlated to target debt ratio (-6.25e-05, p-value less than 0.001). An increase in stock prices, according to financing hierarchy, leads to equity issuance. A negative correlation between leverage and growth confirms the assumptions of market timing theory (managers are reluctant to issue equity when it is under-priced). The regression estimates show a statistically significant and negative correlation between profitability and target leverage ratio (-0.0759, p-value less than 0.001). Highly profitable companies are cash-rich firms which prefer the hierarchy in financing, exploiting retained earnings and internal sources. As for product uniqueness, the regression coefficient of -0.0875 (p-value less than 0.001) demonstrates that unique production and high R&D expenses are mostly related to high growth and profitable companies. Highly levered companies are not able to finance these activities. The difference when compared to previous studies is only found in relation between tangibility and target leverage (-0.0143, p-value less than 0.001). The main explanation for this result can be found in the period covering the analysis, the sixth M&A cycle 2003-2010, which included the global financial crisis. The crisis and post crisis-period were characterized by higher interest rates and tightened credit markets. Moreover, as stated by Lauk (2014), the European Central Bank stimulated short-term funding in the last decade. Companies were largely issuing Commercial Papers (short-term debt), refinancing long-term with short-term debt. Refinancing may happen especially in periods when companies expect a deterioration of cash flows over time (just before and after the crisis). Changing the debt maturity structure is an efficient tool of decreasing the cost of debt and increasing shareholder value. During the recovery period, the growth of the economy is anticipated and stock prices are expected to rise (Drobetz et al., 2007). Therefore, companies are motivated to time the market and issue equity (decreasing leverage),specifically, large and faster growing companies as acquirers, adjust rapidly and have higher financial flexibility in recovery periods. Additionally, from 2000 onwards, classical collateral is no longer sufficient – expected cash flow trends and balance sheet strength became crucially important as collateral (Sherman, 2010). The majority of acquisition from the sample were performed by highly profitable companies. 68 A. MUGOŠA, S. 4.2. Target leverage ratio estimation POPOVIĆ Finally, the heterogeneity of economic development across the countries of Western Europe (e.g. the UK, Germany, and France, compared to Italy, Spain) represents an important factor of capital structure decisions. Capital structure choices are made differently in each country, depending on the level of progress in capital market development, availability of financing sources, corporate governance, legal environment etc. (Xie et al., 2017). European evidence showed that institutional and regulatory framework impact on capital structure policy and decisions over debt issuance (Bancel and Mittoo, 2004). Koralun-Bereźnicka (2017) argues that capital structure in Europe considerably depends on the country in which the company operates, also highlighting the importance of company size and specific industry factors. In line with these results, De Jong et al. (2008) confirmed the importance of firm-specific and country-specific factors as factors of leverage, on a sample of firms on a global level. Kirch et al. (2012) concluded that firm-specific characteristics represent the main factor in the process of capital structure selection. All of these arguments can be a potential cause of tangibility coefficient’s negative sign. 4.3. Estimation of probability of making an acquisition depending on leverage deficit – univariate and multivariate analysis In the second phase, the leverage deficit variable is used in an estimation of the likelihood of making an acquisition. Leverage deficit is defined as the actual leverage ratio minus the estimated target leverage. If the actual leverage ratio is higher than estimated, the company is considered to be overleveraged. However, if actual leverage ratio is lower than estimated, the company is defined as underleveraged. More precisely, firms in the first (fourth) quartile have the lowest (largest) leverage deficit and are defined as underleveraged (overleveraged). Table 3 summarizes leverage deficit descriptive statistics. Even though the mean value of leverage deficit equals zero (on average, actual leverage is equal to target leverage), the large standard deviation of 18.6% implies that the sample consists in companies whose debt deviates from target debt ratio. More precisely, 25% of companies are underleveraged by less than -11.2% (Q1), whilst other, 25% of companies from the sample, are overleveraged by more than 9.8% (Q3). Accordingly, companies tend to change and adjust capital structure in the process of corporate restructuring (acquisitions). These changes are evident in the figure below. DEVIATION FROM TARGET CAPITAL STRUCTURE... 69 DEVIATION FROM TARGET CAPITAL STRUCTURE... DEVIATION FROM TARGET CAPITAL STRUCTURE... 69 Table 3 Leverage deficit descriptive statistics Value Leverage deficit Mean 9.19e-12 Median 0.00062 Standard deviation 0.186 Variance 0.034 Range 3.731 Min -0.540 Max 3.190 Coefficient of variation 2.02e+10 Q1 -0.112 Q3 0.098 Interquartile range 0.210 Source: author’s calculations. -.1 0 .1 .2 2003 2004 2005 2006 2007 2008 2009 2010 Mean Standard deviation Q1 Q3 Fig. 1. Leverage deficit mean, standard deviation, Q1 and Q3 Source: author’s own. Deviations (Q1 and Q3) are present in the whole period of analysis yet Table 3 Leverage deficit descriptive statistics Value Leverage deficit Mean 9.19e-12 Median 0.00062 Standard deviation 0.186 Variance 0.034 Range 3.731 Min -0.540 Max 3.190 Coefficient of variation 2.02e+10 Q1 -0.112 Q3 0.098 Interquartile range 0.210 Source: author’s calculations. Table 3 Leverage deficit descriptive statistics Value Leverage deficit Mean 9.19e-12 Median 0.00062 Standard deviation 0.186 Variance 0.034 Range 3.731 Min -0.540 Max 3.190 Coefficient of variation 2.02e+10 Q1 -0.112 Q3 0.098 Interquartile range 0.210 Source: author’s calculations. Table 3 -.1 0 .1 .2 2003 2004 2005 2006 2007 2008 2009 2010 Mean Standard deviation Q1 Q3 Fig. 1. Leverage deficit mean, standard deviation, Q1 and Q3 Source: author’s own. Fig. 1. Leverage deficit mean, standard deviation, Q1 and Q3 Source: author’s own. Deviations (Q1 and Q3) are present in the whole period of analysis, yet, from 2005, deviations are larger in Q1 (underleveraged group). The mean value of leverage deficit was negative during 2007, 2009 and 2010. It is obvious that large acquirers from the sample have rebalanced their capital 70 A. MUGOŠA, S. POPOVIĆ structure prior to acquisition announcements. Furthermore, in 2007 financial markets’ credit activity was tightened, which additionally impacted on the level of leverage. In line with the probability analysis, it is worth applying both probability analyses, univariate and multivariate. Univariate demonstrates companies’ characteristics by leverage deficit quartiles (by quartiles companies are divided in two groups: underleveraged and overleveraged). The results are shown in Table 4. As quartiles are quantiles that divide data series in four equal categories, the mean values of variables are given in four categories of book leverage deficit: K1 (−∞, Q25], K2 (Q25, Q50], K3 (Q50, Q75], K4 (Q75, +∞). Testing the statistical difference of means was performed applying multivariate tests: Wilks’ lambda, Pillai’s trace, the Lawley-Hotelling trace, and Roy’s largest root. DEVIATION FROM TARGET CAPITAL STRUCTURE... The results confirm that the mean differences from K1- K4 are statistically significant at the 1% level, except for the total return. Table 4 Firm characteristics by book leverage deficit quartiles Leverage deficit quartiles Variables K1 K2 K3 K4 Book leverage 0.375 0.543 0.649 0.801 Tangibility 0.772 0.740 0.747 0.769 Market to book 1.670 1.385 1.279 1.378 Logarithm of sales 9.192 9.303 9.304 9.211 Profitability 0.167 0.148 0.131 0.135 Product uniqueness 0.024 0.021 0.016 0.0192 Total return 24.897 29.069 25.204 27.011 Source: author’s calculations. Table 4 Firm characteristics by book leverage deficit quartiles Comparing the variables from K1 to K4, it can be concluded that companies from K1 are performing better: lower leverage, higher market to book (growth), higher R&D expenses (by 0.5% on average) and higher profitability (by 3% on average). Tangibility and sales values are similar across the categories. On the other hand, the better performance of overleveraged companies is found in the capital market (the return is 2% higher in K4 on average). Even though mean values may potentially imply the higher probability of underleveraged companies to undertake an acquisition, these values do not always change monotonically by leverage deficit categories. This is why multivariate analysis (probit) was applied. In order to contribute to the objectivity of the results, two models using probit methodology were tested. 71 DEVIATION FROM TARGET CAPITAL STRUCTURE... To obtain more precise results, probit model tested the marginal effects of variables (Uysal, 2007, 2011). The marginal effects of continuous variables were computed at the sample means of the data, while the marginal effects of binary variables as the difference between cumulative distribution functions for discrete changes of dummy variables in interval from 0 to 1. The probability of acquisition is related to the success of the undertaken acquisitions. In the case of a successful transaction, the dependent variable takes value 1, and in the case of an unsuccessful transaction, the dependent variable takes value 0. The explanatory variables included in the model are leverage deficit, size, growth and profitability. Previously conducted studies (Datta et al., 2001; Officer,2003; Moeller et al., 2004) showed that big companies have a well- diversified business portfolio, stable cash flows and easy access to sources of financing. Furthermore, acquisition value depends on the acquirer’s and target’s growth perspective (Smith and Kim, 1994). Finally, financial-slack rich bidders will create higher acquisition worth, specifically when acquiring financial slack-poor targets. DEVIATION FROM TARGET CAPITAL STRUCTURE... The model is defined by: ( ) ( ) 0 1 1 1 i P acquistion leveragedeficit Z φ β β β = = + + , (11) (11) where: Zi is vector of explanatory variables and, a φ is cumulative normal distribution function. In order to capture more precisely the effect of leverage deficit, this variable is transformed in dummy variable underleveraged companies, taking the value of 1 for Q1 leverage deficit values, and 0 for all other values. Therefore, the other model is defined as follows: ( ) ( ) 0 1 1 1 i P acquistion underleveraged companies Z φ β β β = = + + , (12) (12) where: Zi is vector of explanatory variables and, a φ is cumulative normal distribution function. The results of the probabilities of both models are given in the following table.ii The first hypothesis is confirmed: the results prove the higher probability of acquisition for the subgroup of underleveraged companies. According to Model 1, the increase of variable underleveraged companies increases the probability of acquisition by 5.42%. A statistically significant and positive relation was found between size, profitability and acquisition probability. It could be concluded that in the region of Western Europe, more success in acquisition process is had by larger and more profitable companies. The variables, growth and average return, have a positive sign but there is no evidence on the statistically significant impact on acquisition probability. 72 72 A. MUGOŠA, S. POPOVIĆ Table 5 Probability of undertaking an acquisition depending on leverage deficit Variable Model 1 Model 2 Underleveraged companies 0.0542*** (0.00905) Market to book (growth) 0.00390 0.00639 (0.00450) (0.00507) Sales (size) 0.0113* 0.0119* (0.00655) (0.00650) Profitability 0.154* 0.170* (0.0578) (0.0595) Total return (growth) 3.86e-05 2.96e-05 (7.08e-05) (7.06e-05) Leverage deficit -0.0248* (0.00850) Number of observations 6,380 6,380 Pseudo R2 0.0054 0.0056 Prob > χ2 0.0000 0.0000 Notes: Standard errors in parentheses/ * p<0.01, ** p<0.05, * p<0.1 Source: author’s calculations. Table 5 A strong negative relation between leverage deficit and acquisition probability was found in Model 2. The increase in leverage deficit by one standard deviation decreases the probability of acquisition by 2.48%, i.e. companies with higher target debt ratios (overleveraged) are less likely to make an acquisition. The coefficients sign and statistical significance of explanatory variables is the same as in Model 1. •• defining the number of observations: 2028, as several bidders undertook more than one acquisition; Source: author’s calculations. 4.4. Estimation of the effects of acquisition announcements on capital markets – CAR estimation The aim of this research stage was to assess the market reaction to the acquisition announcements, measured by bidders’ cumulative abnormal returns. The results of the test answer if there exists a statistically significant relation between CAR and leverage deficit, and if confirmed, is it negative or positive. The test was conducted by applying an event study and consisted of several steps: p •• defining the number of observations: 2028, as several bidders undertook more than one acquisition; •• defining the number of observations: 2028, as several bidders undertook more than one acquisition; 73 DEVIATION FROM TARGET CAPITAL STRUCTURE... •• estimation window was defined in intervals of 250 to 50 days [-250, -50] before the announcement date of acquisitions. The companies without a sufficient number of observations were excluded, and the final number of transactions amounted to 1564; •• estimation window was defined in intervals of 250 to 50 days [-250, -50] before the announcement date of acquisitions. The companies without a sufficient number of observations were excluded, and the final number of transactions amounted to 1564; •• the expected return of each stock was calculated based on the market model. The benchmark return was the value-weighted index of returns including dividends for the index BE500; •• the expected return of each stock was calculated based on the market model. The benchmark return was the value-weighted index of returns including dividends for the index BE500; •• abnormal returns of stocks were calculated as the difference between actual and expected returns;i •• in the five-day event window, two days before and two days after the announ- cement date [-2,2], average abnormal returns and CARs were calculated;ii •• the statistical significance of CARs was confirmed;i •• CARs were regressed over leverage deficit. The relation between capital structure and CARs was estimated with ordinary least square methodology and defined with the following regression equation (Model 1): 0 1 1 2 i i i CAR leveragedeficit size growth β β β β = + + + . (13) 0 1 1 2 i i i CAR leveragedeficit size growth β β β β = + + + . (13) (13) Estimation of Model 1 generated statistically insignificant results, which was the reason to formulate Model 2. In Model 2 variable growth was excluded: 0 1 1 i i CAR leveragedeficit size β β β = + + . (14) (14) The results of both models are given in Table 6. The results of both models are given in Table 6. Table 6 Impact of leverage deficit on CARs Variable Model 1 Model 2 Leverage deficit 0.00333 0.00591 (0.00309) (0.00302) Size -6.63e-11 -2.68e-10 (3.14e-10) (3.08e-10) Growth 1.09e-09 (1.25e-09) Constant 0.00254 0.00162 (0.00361) (0.00327) R2 0.002 0.003 Notes: Standard errors in parentheses/ * p<0.01 ** p<0.05 * p<0.1 Source: author’s calculations. Impact of leverage deficit on CARs 74 A. MUGOŠA, S. POPOVIĆ Model 2 gave statistically significant results of the leverage deficit coefficient. The Link and Ramsey Reset test confirmed that Model 2 was well specified. One can conclude that the leverage deficit is positively correlated with CARs – an increase of leverage deficit by 1% increases CARs by 0.591% (p<0.05). Thus this increase implies that only overleveraged companies undertake the most value-enhancing acquisitions. The value of 0.3% R2 is low, but confirmed in previous research (on average 4%). The low value of R2 can be justified by the lack of data related to the characteristics of transactions which were included in the previous studies. (Moeller et al., 2004; Masulis et al., 2007). Accordingly, the second research hypothesis result is: the market reaction is negative to announcements of underleveraged bidders. The results are consistent with the free cash flow hypothesis (Jensen, 1986), meaning CARs are decreasing when cash-rich bidders are announcing acquisitions, and that capital markets react unfavourably to takeover announcements of underleveraged bidders (Maloney et al., 1993; Uysal, 2007, 2011; Durand et al., 2016; Beschwitz, 2018). CONCLUSION Capital structure adjustments represent a very important factor of the investment decision-making process and company value creation. This paper analysed the impact of deviations from target debt ratio on acquisition choices and market reaction on announcements of these transactions. As noted in the paper, all the results are consistent with previous research focused on the relation between leverage and corporate restructuring. Target debt ratio was regressed on the main determinants widely used: tangibility, growth, size, profitability, product uniqueness and average stock return. The results showed a statistically significant and negative relation of target leverage and each variable, except size. A negative relation between leverage and tangibility is the only result which differs from previous research results. The justification is found in macroeconomic conditions during the period of analysis (the period of the sixth M&A cycle of 2003-2010, which included global financial crises), specific factors of a country in which the company is doing business (the sample is heterogenic – from the UK and Germany, to Italy and Spain), financial market conditions and trends, heterogeneity of companies in the sample, structure of debt (the high proportion of short-term debt leads to a negative relation of leverage and tangibility). Additional arguments were given by Drobetz et al. (2007) in the context of anticipated economy growth in the process of recovery after crisis. The growth 75 DEVIATION FROM TARGET CAPITAL STRUCTURE... motivates companies to issue shares, as their prices are expected to rise. The research confirmed that especially large and fast growing companies (like bidders for companies from the sample) rapidly adjust their capital structure in the recovery period, as these companies are profitable and the majority accumulated internal sources of financing. In the second stage, the probability of making an acquisition was tested by univariate and multivariate (probit) analysis. Both analyses gave the same result: underleveraged companies are more likely to undertake the acquisition. The analysis of two models using probit methodology contributed to the objectivity of the results. Model 1 showed that an increase of dummy variable unleveraged companies by one standard deviation increased the probability of acquisition by 5.42%, while Model 2 tested the probability depending on leverage deficit, and the results showed that an increase of leverage deficit by one standard deviation decreased the probability of acquisition by 2.48%. REFERENCES Ahmed, Y., Elshandidy, T., Why Do Over-Deviated Firms from Target Leverage Undertake Foreign Acquisitions?, “International Business Review“, Vol. 27, No 2, pp. 309-327, 2018. Andrade, G., Mitchell, M., Stafford, E., New Evidence and Perspectives on Mergers, “Journal of Economic Perspectives“, Vol.15, No 2, pp. 103-120, 2001. Baker, H. K., Martin, G. S., Capital Structure and Corporate Financing Decisions. The Robert W. Kolb Series in Finance. John Wiley & Sons, Inc., Hoboken, New Jersey, 2011. Baker, M., Wurgler, J., Market Timing and Capital Structure, “Journal of Finance“, Vol. 57, No 1, pp. 1-32, 2002. 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M., Roll, R., The Adjustment of Stock Prices to New Information, “International Economic Review“, Vol. 10, No 1, pp. 1–21, 1969. Fama, E., French, K. CONCLUSION An increase of leverage deficit is a positive deviation from target debt ratio (overleveraged companies), which confirms that underleveraged companies have a higher probability to engage in acquisitions. i In the final stage, the test of CARs indicated that the market reacts unfavourably to acquisition announcements of underleveraged companies, which is consistent with Jensen’s free cash flow hypothesis. If leverage deficit increases by 1%, CARs will increase by 0.591%. Consequently this increase implies that only overleveraged companies undertake the most value- enhancing acquisitions and that underleveraged companies make poor acquisition choices. i A negative market reaction is confirmed in various papers analysing markets globally. Yet, only Uysal (2007, 2011) applied the concept of leverage deficit in the context of acquisition decisions in the US market. This research contributes to the analysis of the leverage deficit concept, as capital structure shaping represents an actual scientific dilemma not widely researched in Western Europe. The empirical verification of the model could be beneficial for managers in the process of evidence-based capital structure decision- making. This paper provides the findings on the most important factors of optimal capital structure, as well as on the impact of this structure on future investment decisions. Finally, the research opens up the possibility to compare findings and test differences between the EU and US capital markets. The continuous changes of capital structure in the global market (high leverage) imply the necessity of a deeper analysis of M&A financing and the success of these transaction. 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https://openalex.org/W4390474997	https://waraqat.assunnah.ac.id/index.php/alhimam/article/download/590/252	Arabic	null	Tahlil Asbab Do'fi Qudrati al Tollab 'Ala Tathbiq Maharah Kalam Fi Madrasah al Imam al Muslim	Al Himam	2,023	cc-by	4,622	Abdur Rahman Purba1, Arief Wijaksono2, Achmad Farhan Rafli3 Abdur Rahman Purba1, Arief Wijaksono2, Achmad Farhan Rafli3 1Sekolah Tinggi Ilmu Tarbiyah (STIT) Ar-Raudhatul Hasanah, Medan 2,3Sekolah Tinggi Agama Islam As-Sunnah, Deli Serdang ariefwijaksono0908@gmail.com 1 2 S k l h Ti i A I l A S h D li S 2 Vina Anisatul Fauji, Darul Qutni, and Muchlisin Nawawi, “Efektivitas Media Flashcard Terhadap Kemampuan Membaca Nyaring (Al-Jahriyah) Dan Membaca Dalam Hati (Ash-Shamitah) Bahasa Arab Siswa Kelas Viii Mts Negeri 1 Purbalingga Tahun Ajaran 2018/2019,” Lisanul Arab: Journal of Arabic Learning and Teaching 9, no. 1 (June 29, 2020): 74–79, https://doi.org/10.15294/la.v9i1.39311. 1 Ade Muhammad Ritonga, Danang Prasetio, and Fakhrurrozi Fakhrurrozi, “مح ةيمالسإلا نيملعملا ةيلك ريدم تالوا في ىدل ةيبرعلا ةغللا ةيقرت ةيلامشلا ةرطموس هاجنوت يناب رثوكلا دهعمب صصختملا لوألا فصلا يف بالطلا,” Lahjah Arabiyah: Jurnal Bahasa Arab Dan Pendidikan Bahasa Arab 3, no. 2 (June 23, 2022): 91–101, https://doi.org/10.35316/lahjah.v3i2.91-101. اصخلمل اصخلمل ص ال ثحبلا اذه نم فدهلع ةفرعمل ةقيش ةشقانم هذه نأ اهدحأ ،ثحبلا عقوم يف ليلحت ءارجإ دعب لماوع ةد سب تاردق روطتت ىتح اهلح ةيفيكو ب ةسردملا يف بالطلا.مدختسملا جهنملاو يه ملا يفصولا يفيكلا جهن وا قرطل ال ليلحتلا قرط مادختساب تانايبلا ليلحتل امنيب ، قيثوتلا قرطو ةلباقملاو ةظحالملا يه تانايبلا عمجل ةمدختسم يفصولا،نأ يه ثحبلا ةجيتنو أس دوجو مدع : ةغللاب ثدحتلا ىلع بالطلا ةردق فعض باب يبرعلا ملعم، عد م ةيوغللا ةئيبلا، مدع ةيبالطلا ةمظنملا ءاغلإ دعب ةيبرعلا ةغللا قيبطتل ةبقارملا خاص ،ةغللا مسق ةقل طاشن ة تادرفملا ظفح لثم لصفلا جراخ يوغل ت ،ةيوغللا ةطشنألا نم ليلق ، ةيبرعلا ةغللا جهنملا رييغ، بح نم ليلقلا الط ،ةيبرعلا ةغلل بال برعلا ةغللا ةسرمملا ةلقية ،ةسردملا ةئيبلافأم لحك ةيوغللا تاراهملا ريوطت ةيفيك نع ا لتح :يهف ،ةسردمل ا يف ةب سانملا ة يوغللا بالطل ا تاراهم نيس ةعانص بي ةئ لالغ تاودن ءاطعإو ،ةيو لال ةيوغ لل ةطشنأ لمعو ، ةفثكم لوصف لمعو ،نيملعم لالةيوغ الك :ةيسيئرلا ةملا ؛بابسأل بالطلا فعض ؛ ممالكلا ةراه AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|33 ةمدقملا ال ركفلا نع ريبعتلا ةدأ يه ةغلو ال يتلا تاراشإلاو زومريست ،رشبلا عم لصاوتلا فدهب ناسنإلا اهمدخ ورعاشم نع ريبعتلا عمتجملا لخاد سانلا نيب ميهافتلا لئاسو ىدحإ ةغللا دعتو ،ةفرعملا باشتكاو ،ه. 1 تت رصانع ةثالثو تاراهم عبرأ نم ةيبرعلا ةغللا نوكفتاراهم عبرأ هراهم :ية اراهمو ، عامتسال ة لا مالك، 2 ورعاشم نع ريبعتلا عمتجملا لخاد سانلا نيب ميهافتلا لئاسو ىدحإ ةغللا دعتو ،ةفرعملا باشتكاو ،ه. تت رصانع ةثالثو تاراهم عبرأ نم ةيبرعلا ةغللا نوكفتاراهم عبرأ هراهم :ية اراهمو ، عامتسال ة لا مالك، وراهمة الق ،ةءاروراهمة ا،ةباتكل 2 وةث الث عرصان ا : اهلأصاو ،تاو ملفتلاو ،تادرر او .بيك هذه رصانعلا يه امل ا ل ا ةد لا ةيقيقح يت ني عت امل لع ملعت ى تع نمو ،ةغللا تاراهم مل ل رطيسي م عل اهيلا يملا نم نكمتسي رط ى لع ب ةغللا تاراهم مسيوتاملا ا هتتةددع ى لع ب ةغللا تاراهم مسيوتاملا ا هتتةددع مه ةغللا يف ةمهم ةراهم يه مالكلا ةراال مهألا نمو ،ضعب مهضعب مهلاصتالل نوملكتي عمتجملا نأل ةيبرع عن ةغللا ملعم هكلسي نأ وجرن يذلا هاجتالا وه اذهو يهفشلا بناجلاب مامتهالا وه ةيبرعلا ةغللا ميلعت د ال نييالم اهمهفي ،لاصتا ةغل ةيبرعلا نأل ةيبرعلاب ثيدحلا نم بالطلا نيكمت لوألا همه لعجي نأو ،ةيبرع نلا ملاعلا يف سا. (J ) p g 3 Ibnu Fisabilillah, Tiy Kusmarrabbi Karo, and Ilham Tumanggor, “Isti’ab al-Mufrodat Wa ’Alaqotuha Binatijah at-Thallab Fi Hifdzi al-Quran,” Al-Himam: Jurnal Ilmu Ilmu Pendidikan & Bahasa Arab 2, no. 1 (December 20, 2022): 51–64, https://doi.org/10.51590/alhimam.v1i1.366. AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|34 4 Acep Hermawan, Metodologi Pembelajaran Bahasa Arab (Bandung: PT Rosda Karya, 2013). h. 72 1 Ade Muhammad Ritonga, Danang Prasetio, and Fakhrurrozi Fakhrurrozi, “مح ةيمالسإلا نيملعملا ةيلك ريدم تالوا في ىدل ةيبرعلا ةغللا ةيقرت ةيلامشلا ةرطموس هاجنوت يناب رثوكلا دهعمب صصختملا لوألا فصلا يف بالطلا,” Lahjah Arabiyah: Jurnal Bahasa Arab Dan Pendidikan Bahasa Arab 3, no. 2 (June 23, 2022): 91–101, https://doi.org/10.35316/lahjah.v3i2.91-101. 2 Vina Anisatul Fauji, Darul Qutni, and Muchlisin Nawawi, “Efektivitas Media Flashcard Terhadap Kemampuan Membaca Nyaring (Al-Jahriyah) Dan Membaca Dalam Hati (Ash-Shamitah) Bahasa Arab Siswa Kelas Viii Mts Negeri 1 Purbalingga Tahun Ajaran 2018/2019,” Lisanul Arab: Journal of Arabic Learning and Teaching 9, no. 1 (June 29, 2020): 74–79, https://doi.org/10.15294/la.v9i1.39311. 3 Ibnu Fisabilillah, Tiy Kusmarrabbi Karo, and Ilham Tumanggor, “Isti’ab al-Mufrodat Wa ’Alaqotuha Binatijah at-Thallab Fi Hifdzi al-Quran,” Al-Himam: Jurnal Ilmu Ilmu Pendidikan & Bahasa Arab 2, no. 1 (December 20, 2022): 51–64, https://doi.org/10.51590/alhimam.v1i1.366. 4 Acep Hermawan, Metodologi Pembelajaran Bahasa Arab (Bandung: PT Rosda Karya, 2013). h. 72 5 Anggie Sri Utari, “Atsar Istikhdam Wasilah Dam’iyah al Yad Fi Ta’lim Maharoh al Kalam Bil Madrosah al Ibtidaiyah,” Al-Himam: Jurnal Ilmu Ilmu Pendidikan & Bahasa Arab 2, no. 2 (June 28, 2023): 49–64, https://doi.org/10.51590/alhimam.v2i2.450. 7 Nandang Sarip Hidayat, “اهميلعت ةءارجإو مالكلا ةراهم,” Al-Manar 2, no. 8 (2018), https://doi.org/10.24014/al- manar.v2i8.4736. p // g/ / 6 Muhammad Evan Alfian, “Keterampilan Berbicara Dan Pengajarannya,”Arabia ,Jurnal Pendidikan Bahasa Arab 5, no. 1 (2014): 103–21. AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|35 5 Anggie Sri Utari, “Atsar Istikhdam Wasilah Dam’iyah al Yad Fi Ta’lim Maharoh al Kalam Bil Madrosah al Ibtidaiyah,” Al-Himam: Jurnal Ilmu Ilmu Pendidikan & Bahasa Arab 2, no. 2 (June 28, 2023): 49–64, https://doi.org/10.51590/alhimam.v2i2.450. 6 Muhammad Evan Alfian, “Keterampilan Berbicara Dan Pengajarannya,”Arabia ,Jurnal Pendidikan Bahasa Arab 5, no. 1 (2014): 103–21. 7 Nandang Sarip Hidayat, “اهميلعت ةءارجإو مالكلا ةراهم,” Al-Manar 2, no. 8 (2018), https://doi.org/10.24014/al- اصخلمل 4 ةراهم ال يف .ةيبنجأ ةغلب اهناقتإ يف بالطلا بغري يتلا ةيساسألا تاراهملا دحأ وه مالك تع ةيبرعلا ةغللا وسردم هيطعي نأ لمأن ءيش اذهو ، قوطنملا بناجلا ىلإ هبتنن نأ بجي ، ةيبرعلا ةغللا ميل 3 Ibnu Fisabilillah, Tiy Kusmarrabbi Karo, and Ilham Tumanggor, “Isti’ab al-Mufrodat Wa ’Alaqotuha Binatijah at-Thallab Fi Hifdzi al-Quran,” Al-Himam: Jurnal Ilmu Ilmu Pendidikan & Bahasa Arab 2, no. 1 (December 20, 2022): 51–64, https://doi.org/10.51590/alhimam.v1i1.366. ال.ةيولوأ يس اهمهفي يتلا لاصتالا ةغل يه ةيبرعلا ةغللا نأل ةيبرعلا ةغللاب ثدحتلاب بالطلل اذه حم مع ريغ ةيظفللا ةغللا نأب قوطنملا بناجلا نولهاجتي نيذلا صاخشألل ةجح دجوت ال ،نيملسملا مظ م طقف سيل اهملكتي دحأ الو ةدوجو ةغللا ةبوتكملا. كلا ةيمنت نإلا ا وأ م لحمو بسانم جمانرب دادعإ مزلتست راو خ فادهأ قيقحتل طط محد نم ةدمتسم ةد ا سسأ ل ف ةيبرعلا ةغللا ميلعت فادهأ نمو لاعفلا راوح ي ب ,ةنيعم ةيسارد ةلحرمحلا لمتشي ثي برن جما ا ةغايص ىلعلأ ملا ميظنتو رايتخاو فاده حتو ىو ت إلاو سيردتلا قرط ديدح ةيميلعتلا لئاسولاو ةطشن . يوقتلا بيلاسأو م ملا ةبسان.ا و لأسملا بيلا ف ةمدختس ي لاو ملاكلا سيردت ح يلي ام يهف راو؛ 1 ) ال ةثداحم ةهجوملا، 2باعلألا )، 3ةشقانملا )، 4 ) ةريغصلا تاعومجملا ةيجيتارتسا. 6 أمالكلا ةراهم ميلعت فاده إن يف مالكلا ةراهم لا مف ،مهم رومألا نم ةيبنجألا ةغلن مهملا رومألا نم فادهأة هذ ؛ يلي امك ه1 ) نطق الأ احيحص اقطن ةيبرعلا تاوص، 2 ) ب قطنلا دنع زييمتلا ني الأ ا تاوص لم باشت م ت ة ه احضاو ازيي، 3 ) ال زييمت ب قطنلا دنع ني ح لا صقلا تاكرير ةليوطلاو ة، 4 ) بنلا عاونأ ةيدأتر وا يثدحتم نم ةلوبقم ةقيرطب ميغنتل ةيبرعلا، 5 ) نط قاأل ملا تاوصت احيحص اقطن ةرواج( مثل ...ث ,ت ,ب لخ إ(ةدايزو ،. 7 7 Nandang Sarip Hidayat, “اهميلعت ةءارجإو مالكلا ةراهم,” Al-Manar 2, no. 8 (2018), https://doi.org/10.24014/a .v2i8.4736. 7 Nandang Sarip Hidayat, “اهميلعت ةءارجإو مالكلا ةراهم,” Al-Manar 2, no. 8 (2018), https://doi.org/10.24014/al- manar.v2i8.4736. اصخلمل قر ميلعتلا تالكشمو ،ةيبنجألا ةغللب مالكلا ةيفعضب قلعتي ردصملا ضعب ثحابلا أال يف ةيبرعمه ةرا مالكلا، ام حرشي أسباب اببسي يذل عضو ف، ،مالكلا قيبطت دنع بالطلا زجع ببسي لماوعلا امو و كي ف يكو ،لئاسولا يأو ،سيردتلا نو لاطر أرقو .اضعب مهضعب رثأي اهلك ،لصفلا يف ةلمعتسملا ةقي حبلا نع ثحابلاو ث عاونأب ةهبشملا ال" عوضوم حتلي ىدل ةيوناثلا ةلحرملا يف مالكلا ةراهم ميلعت تالكشم ل ةيرمقلا يرصعلا دهعمب يناثلا فصلا بالط غغنلا 8" عئاش رمأ ،مالكلا ةراهم رمأ يف تالكشملا نأ ىلع لد، ل،ةرركتم تالكشملا اذام وأ" عوضوملاب ثحبلا كلاذكسب ىدل ةيبرعلا ةغللاب مالكلا ةراهم فعض باطل با ايوونلا مامإلا دهعملاب ةطسوتملا ةلحرم يف يناثلا فصل" 9 وأي" عوضوملاب ثحبلا اضمش ةراهم ميلعت تالك ااوغورف نولوك ستاو ةيناثلا ةيموكحلا ةيمالسإلا ةيوناثلا ةسردملا يف مالكل 10 " وىلع اسسأ هذ دارأ ا ال ثحبلا ثحابعن أس ةغللاب ثدحتلا ىلع بالطلا ةردق فعض بابال ىدل ملسم مامإلا دهعمب ةيبرع الوألا لصفلا ةيوناثلا ةلحرملا يف بالطل. ال ناكم يف الوأ ثحابلا ثحبلاب روكذملا ثوحبلا نيب قرف وا اذه يل نأ ،عوضوملت.دقف اهتراهم يف سيلو ةردقلا ةملكلاب ديدح ةفرعمل ثحبلا اذه نم درغلا أبابس ةيبرعلا ةغللاب ثدحتلا ىف بالطلا ةردق فعض و ةفرعمل طر ةغللاب ثدحتلا ىلع بالطلا ةردق ريوطت ةقي ةيبرعلا. قر ميلعتلا تالكشمو ،ةيبنجألا ةغللب مالكلا ةيفعضب قلعتي ردصملا ضعب ثحابلا أال يف ةيبرعمه ةرا مالكلا، ام حرشي أسباب اببسي يذل عضو ف، ،مالكلا قيبطت دنع بالطلا زجع ببسي لماوعلا امو و كي ف يكو ،لئاسولا يأو ،سيردتلا نو لاطر أرقو .اضعب مهضعب رثأي اهلك ،لصفلا يف ةلمعتسملا ةقي حبلا نع ثحابلاو ث عاونأب ةهبشملا ال" عوضوم حتلي ىدل ةيوناثلا ةلحرملا يف مالكلا ةراهم ميلعت تالكشم ل ةيرمقلا يرصعلا دهعمب يناثلا فصلا بالط غغنلا 8" عئاش رمأ ،مالكلا ةراهم رمأ يف تالكشملا نأ ىلع لد، ل،ةرركتم تالكشملا اذام وأ" عوضوملاب ثحبلا كلاذكسب ىدل ةيبرعلا ةغللاب مالكلا ةراهم فعض باطل با ايوونلا مامإلا دهعملاب ةطسوتملا ةلحرم يف يناثلا فصل" 9 وأي" عوضوملاب ثحبلا اضمش ةراهم ميلعت تالك ااوغورف نولوك ستاو ةيناثلا ةيموكحلا ةيمالسإلا ةيوناثلا ةسردملا يف مالكل 10 " وىلع اسسأ هذ دارأ ا ال ثحبلا ثحابعن أس ةغللاب ثدحتلا ىلع بالطلا ةردق فعض بابال ىدل ملسم مامإلا دهعمب ةيبرع الوألا لصفلا ةيوناثلا ةلحرملا يف بالطل. ال ناكم يف الوأ ثحابلا ثحبلاب روكذملا ثوحبلا نيب قرف وا اذه يل نأ ،عوضوملت.دقف اهتراهم يف سيلو ةردقلا ةملكلاب ديدح ةفرعمل ثحبلا اذه نم درغلا أبابس ةيبرعلا ةغللاب ثدحتلا ىف بالطلا ةردق فعض و ةفرعمل طر ةغللاب ثدحتلا ىلع بالطلا ةردق ريوطت ةقي ةيبرعلا. 11 Abdul Wahab Rosyidi dan Mamlu’atul Ni’mah, Pembelajaran Bahasa Arab (Malang: UINMALIKI Press, 2). h. 88 اصخلمل AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|36 8 حسب ,راغيرس قدصلا يت ةيرمقلا يرصعلا دهعمب يناثلا فصلا بالط ىدل ةيوناثلا ةلحرملا يف مالكلا ةراهم ميلعت تالكشم ليلج غنلاغ (د ,جنادرس يلي2022 ). 9 ه مامإلا دهعملاب ةطسوتملا ةلحرم يف يناثلا فصلا بالط ىدل ةيبرعلا ةغللاب مالكلا ةراهم فعض بابسأ“ ,نويتسان ايلوج سن الن ,ةيمالسإلا ةنسلا ةعماج( ”يوو2021 ). 10 ظ ةنسلا ةعماج( ”اوغورف نولوك ستاو ةيناثلا ةيموكحلا ةيمالسإلا ةيوناثلا ةسردملا يف مالكلا ةراهم ميلعت تالكشم“ ,اويكبس رها ال ,ةيمالسإ2013 ). AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|36 ال ةمدختسملا ةقيرطهولا ب ثحا جهنمب يفيكلا،يليلحتل وا ةرش ابم ثحابلا ىري نأ تانيبلا عمج ةقيرطل إ ل( ثح بلا ن اكم ى ةظحالم)، ث يلثتلا ب تانيبل ا دكؤيو (م،)اهريغو ةلباقم ،ةظحال وا يذلا تا وطخل سي الوأ : يه ثحابلا هلمعتس كلذ ريغ وأ ةيلعافتلا ةيرظنلا قيرط نع امإ تانايبلا عمج، كلذ دعبو تب تانايبلا رايتخا يأ ،تانايبلا طيس عمجو ثحبلا ضارغأل ةبسانملا تانايبلا، مثجع تانايبلا طيسبت ل ثحبلا ضارغأل ةبسانملا تانايبلا عمجو رايتخا يأ ، الماك ثحبلا نوكي ىتح ،. ال ةمدختسملا ةقيرطهولا ب ثحا جهنمب يفيكلا،يليلحتل وا ةرش ابم ثحابلا ىري نأ تانيبلا عمج ةقيرطل إ ل( ثح بلا ن اكم ى ةظحالم)، ث يلثتلا ب تانيبل ا دكؤيو (م،)اهريغو ةلباقم ،ةظحال وا يذلا تا وطخل سي الوأ : يه ثحابلا هلمعتس كلذ ريغ وأ ةيلعافتلا ةيرظنلا قيرط نع امإ تانايبلا عمج، كلذ دعبو تب تانايبلا رايتخا يأ ،تانايبلا طيس عمجو ثحبلا ضارغأل ةبسانملا تانايبلا، مثجع تانايبلا طيسبت ل ثحبلا ضارغأل ةبسانملا تانايبلا عمجو رايتخا يأ ، الماك ثحبلا نوكي ىتح ،. AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \| AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|37 12 Agus Setyonegoro, “Hakikat, Alasan, Dan Tujuan Berbicara, Dasar Pembangun Kemampuan Berbicara Mahasiswa,” Jurnal Pendidikan Bahasa Dan Sastra Vol.3 (2013): No.1. h. 76 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|38 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|38 نةشقانملاو ثحبلا جئات ل ساردلا ةراهملا نم ءزج وه مالكلا نأل .ةغللا يف ةراهملا مهأ يه مالكلا ةراهم ية ةراهم ريبعت كلذلف ،بالطلل ةيبنجألا ةغللا ميلعت يف ساسأ ءزج مالكلا 11 . ربتعت .ةغللا يف ةراهم رثكألا يه ثدحتلا ةراهممه ثدحتلا ةرا جز نود رمتسم ل كشب رارمتساب ثدحتلا ةسرامم يه مالكلا ةراهم .ةيبنجألا ةغللا ملعت نم اًدج يساسأ ء يتوصلا ريبعتلا مادختساب تادرفملا سفن راركت نودبو فقوت. بع ءاهتنالا دال ثحابمن اهددحي ةرتف لالخ ثحبلا عىل أس ىدل ةيبرعلا ةغللاب ثدحت ةراهم فعض باب ةيوناثلا ةسردم بالط نم لوألا فصلا بالط يلي اميف؛ ساردلا ةراهملا نم ءزج وه مالكلا نأل .ةغللا يف ةراهملا مهأ يه مالكلا ةراهم ية ةراهم ريبعت كلذلف ،بالطلل ةيبنجألا ةغللا ميلعت يف ساسأ ءزج مالكلا 11 . ربتعت .ةغللا يف ةراهم رثكألا يه ثدحتلا ةراهممه ثدحتلا ةرا جز نود رمتسم ل كشب رارمتساب ثدحتلا ةسرامم يه مالكلا ةراهم .ةيبنجألا ةغللا ملعت نم اًدج يساسأ ء يتوصلا ريبعتلا مادختساب تادرفملا سفن راركت نودبو فقوت. بع ءاهتنالا دال ثحابمن اهددحي ةرتف لالخ ثحبلا عىل أس ىدل ةيبرعلا ةغللاب ثدحت ةراهم فعض باب ةيوناثلا ةسردم بالط نم لوألا فصلا بالط يلي اميف؛ 1. ةلق ملا يبرعلا ملع ؛ لعت نم فادهأ ي ق بالطلا نوكي نأ وه مالكلا م نيردا عل لكشب اًيظفل لصاوتلا ى صح يتلا ةغللا مادختساب يعيبطو حي تمك ةسرامم نم فادهأ ،ماع لكشب .بطاخملا اهمهفي نأ ن ال نكمتي نأ وه ئدتبملا ىوتسملل ملكت ةيمويلا ةايحلا يف ةطيسب ةقيرطب اًيظفل لصاوتلا نم بالطلا، ينعي يرشبلا مالكلل فادهأ ةدع كانه ؛ ( )أ ءارآلاو لايخلاو رعاشملاو راكفألا نع ريبعتلا، ( )بتج بوا 11 Abdul Wahab Rosyidi dan Mamlu’atul Ni’mah, Pembelajaran Bahasa Arab (Malang: UINMALIKI Press, 22). h. 88 1122). h. 88 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|37 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|37 مع معنى املحادثة من اآلخرين (ج ) نيرخآلا نع هيفرتلا ديرت، )د( تامولعملا لاسرإ، )ه(إق نيرخآلا عان مهيلع ريثأتلا وأ12. 13 Noza Aflisia and Partomuan Harahap, “Eksisten Bi’ah Lughawiyah Sebagai Media Berbahasa Arab Dalam Meningkatkan Kemampuan Muhadatsah Mahasiswa Prodi Pendidikan Bahasa Arab IAIN Curup,” Lisanul’Arab: Journal of Arabic Learning and Teaching 8, no. 1 (2019): 40–55. مع معنى املحادثة من اآلخرين (ج ) نيرخآلا نع هيفرتلا ديرت، )د( تامولعملا لاسرإ، )ه(إق نيرخآلا عان مهيلع ريثأتلا وأ12. يم بجي ، ةغللا ملعت يف ،سانلا ىدل ةغللا نيوكت يف حاجنلا حيتافم دحأ مه نيثدحتملا نأ ملعن نأ اننك أن يتلا ةغللا مادختساب صاخشألا ءالؤه عم لصاوتلا ىلع نورداق صاخشأ وأ ةعومجم كانه نوكت ير ةذتاسألا ضعب كانه ،ملسم مامإلا دهعم يف .اهملعت نوديال ةيبرعلا ةغللاب ثدحتلا ىلع نيرداق اًيباتكو اًيهفش، ووديري الو ةذتاسألا ضعب نم نأ فسألا نمف ، كلذ عمن أن.ةيبرعلا ةغللاب اوثدحتي ارود هدنع سردمل مهًم لاثم اونوكي نأ بجيو ، ةغللا نيوكت يف نيي يح نوثدحتي نيذلا مهبالطل هب ىذت ا مهئالمز ىلع اهقيبطتو ةيوغللا مهتاراهم ديلقت بالطلل نكمي ةقيرطلا هذهب كلذ عمو ،اًمئاد ةيبرعل ال بالطمع ال ال نارصنع امه مالكلاو عامتسإلا ةراهملا نأ انفرع دقو .سردملا عم بالطلا وأ بالط ين نيذلا صاخشألا مهو ، نيترهاملا نيتاه يف مهم رود مهدنع نوثدحتملا ، ةلاحلا هذه يف ،نالصف يت ةغللا ىلإ عامتسالا ىلع مهديوعت لالخ نم بالطلا نودوؤي نيذلا صاخشألاو ةيبرعلا نوثدح ارعلبي .مهناذآ ىلع ةبيرغ ةيبرعلا ةغللا نوكت ال ىتح ،ة ف نيثدحتم دوجو مدع وه ملسم مامإلا دهعملا يف نيثدحتملا ىدل روصقلا هجوأ دحأ نإ اهب نيقطان. 2. ةيوغللا ةئيبلا مدع ؛ ال ةرود اهدنع ةئيبلا .سانلا نم ةعومجم وأ صخش هيف شيعي ناكم يه ةئيب مه ةغللاب ثدحتلا مزلي ثيح ةغللا نيوكت يف ةيلعاف رثكأ نكسلا اهب يتلا تائيبلا .ةغللا نيوكت يف ةًم 2. ةيوغللا ةئيبلا مدع ؛ ال ةرود اهدنع ةئيبلا .سانلا نم ةعومجم وأ صخش هيف شيعي ناكم يه ةئيب مه ةغللاب ثدحتلا مزلي ثيح ةغللا نيوكت يف ةيلعاف رثكأ نكسلا اهب يتلا تائيبلا .ةغللا نيوكت يف ةًم العربية فقط، 13 في ةيوغللا ةئيبلا ةايحل لقأ اًمامتها يلوت ملسم مامإلا دهعم نأ ودبي ، ةلاحلا هذه في بالطلا ةعباتم يف ةمراص تابوقع وأ ةمراص ةمظنأ دوجو مدع ىلع ليلد دوجو عم دهعملا كلذ ا.ةغللا نوفلاخي نيذل ذل بالطلا نم ريبكلا ددعلاو ،ةغللا يفلاخمل ىوصقلا ةبوقعلا مدع نم ك ال مهتبقاعم نأشب قلقلا نود ملسم مامإلا دهعملا ةئيب يف ةينالع ةيسينودنإلا ةغللاب نوثدحتي نيذ لنكسلا ةئيب يف ةغللا دعاوق مهكاهتنا ، ثم ريوطت نع ةلوؤسملا ةيوغللا ةنجللا لاغتشا مدع كلذ عبت ا.ملسم مامإلا ةسردم يف ةيوغللا ةئيبلا ضرفو ةيوغللا داومل 3. AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|39 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|39 14 Amel Benarioua, 14 Amel Benarioua, “ةيمالعالا ةغللا ةسراممو ةيبرعلا ةغللا نيب مالعالا بلاط,” Human Sciences Journal, October 3, 2021, –604 وا ةيبرعلا باتك اوسرد نأ مهل قبس ثيح ةيبرعلا ةغللا يف ةيفلخ مهيدل نيملعملا ءالؤه نأ وه ببسل ب.باتكلا نم ةدام سيردت يف اًكبترم ناك هنأ اوفرتعا يذلاو ، كيدي ني وم نولذبي اولاز ام ، كلذ ع قص.مهبالطل لضفألا ميدقتل مهدهج ىرا وا ةيبرعلا باتك اوسرد نأ مهل قبس ثيح ةيبرعلا ةغللا يف ةيفلخ مهيدل نيملعملا ءالؤه نأ وه ببسل ب.باتكلا نم ةدام سيردت يف اًكبترم ناك هنأ اوفرتعا يذلاو ، كيدي ني وم نولذبي اولاز ام ، كلذ ع قص.مهبالطل لضفألا ميدقتل مهدهج ىرا 6. AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|40 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|40 مع معنى املحادثة من اآلخرين (ج ) نيرخآلا نع هيفرتلا ديرت، )د( تامولعملا لاسرإ، )ه(إق نيرخآلا عان مهيلع ريثأتلا وأ12. عد م ةيبرعلا ةغللا قيبطتل ةبقارملا، كا ملسم مامإلا دهعم ىدل ن ملانظ بالط اهريدت ةيبالطلا ةم بع دهمإما ىمست ملسم مOSPIM ( م)ملسم مامإ ةيبالط ةمظن، ةطشنألا ىلع ةبلطلا ربدت ةمظنملا ملا ىغلإب نيرخآلا هيجوت يف ةبلطلا رود كانه نوكي ال نامز دعبو .ةعونتملا بالطلا ةمظنة ل دقف نإف اذ ةيلاثم لقأو فعضأ ةغللا ةئيبل ىصقألا نيوكتلا لعجي ميظنتلا اذه. غم 3. عد م ةيبرعلا ةغللا قيبطتل ةبقارملا، كا ملسم مامإلا دهعم ىدل ن ملانظ بالط اهريدت ةيبالطلا ةم بع دهمإما ىمست ملسم مOSPIM ( م)ملسم مامإ ةيبالط ةمظن، ةطشنألا ىلع ةبلطلا ربدت ةمظنملا ملا ىغلإب نيرخآلا هيجوت يف ةبلطلا رود كانه نوكي ال نامز دعبو .ةعونتملا بالطلا ةمظنة ل دقف نإف اذ ةيلاثم لقأو فعضأ ةغللا ةئيبل ىصقألا نيوكتلا لعجي ميظنتلا اذه. ي ا أ 4. لصفلا جراخ يوغل طاشن ةلق؛ مو تادرفملا ظفح يوغللا ةطشنألا لهسأ نتع رغصأ نم تادرفملا د عنا .ةغللا تادرفم يهو ةغللا رص عقاولا لا هيف نوكي أن ةطشخا( ملعتلاو سيردتلا ةطشنأ جرKBM ). 5. ت جهنم رييغ دةسار ةيبرعلا ةغللا ؛ ال لقن يف ملعملا حاجنب اًقيثو اًطابترا طبت رت ةادأ يه سردتلا بتك ال ةقيرطو ةقيرط ىلع ديكأتلاب ملعملا تافصاوم عم قفاوتت يتلا ةيسردملا بتكلا رثؤتس .هبالط ىلإ داوم ت.بلاطلا ىلإ ملعملا نم داوملا ليصو ا باتكلا يسردمل ال ةسردملاب لوألا فصلا بالط همدختسا يذ غللا سورد باتك وه ايلعلا ة ال نم ىواكش كانه تناك ةقباسلا ةلباقملا ةلحرم يف .روطنغ نم ةيبرع ال.باتكلا مادختساب ةيبرعلا ةغللا سيردت يف تابوعص وأ لكاشم اودجو نيذلا نيملعملا نم ديدع 13 Noza Aflisia and Partomuan Harahap, “Eksisten Bi’ah Lughawiyah Sebagai Media Berbahasa Arab Dalam Meningkatkan Kemampuan Muhadatsah Mahasiswa Prodi Pendidikan Bahasa Arab IAIN Curup,” Lisanul’Arab: Journal of Arabic Learning and Teaching 8, no. 1 (2019): 40–55. وا ةيبرعلا باتك اوسرد نأ مهل قبس ثيح ةيبرعلا ةغللا يف ةيفلخ مهيدل نيملعملا ءالؤه نأ وه ببسل ب.باتكلا نم ةدام سيردت يف اًكبترم ناك هنأ اوفرتعا يذلاو ، كيدي ني وم نولذبي اولاز ام ، كلذ ع قص.مهبالطل لضفألا ميدقتل مهدهج ىرا قةيبرعلا ةغللا ةسارد يف بالطلا ةبغر ةل ؛ سي ةغلل بالطلا ةبغرلا وأ بح ةلق يدؤا ىلإ ةيبرعل ان ةغللاب ثدحتلا ىلع نيرداق اونوكي نأ يف بالطلا نم اريثك بغري ثيح .اهب نيثدحتملا ددع ضافخ ا.ةيبرعلا ةغللاب ثدحتلاب مهتغل ةسرامم نوديري ال مهنكلو ةيبرعل حيث ، ءاقدصألا وأ نيسردملا ءاوس ، مهلوح نم صاخشألا عم اًريثك نولصاوتي بالطلا لازي ال ثدحتي بلل ةيبرعلا ةغللاب ثدحتلل تاريذحتلاو عفادلا اوقلت مهنأ نم مغرلا ىلع ، ةيسينودنإلا ةغ د.اًمئا قلم ةما ةسردملا ةئيب يف ةيبرعلا ةغللا ةسر ؛ هذ وأ ذيفنتل صخشلا ةلواحم يه ةسرامملا ه ت.ةيبرعلا ةغللا ملعت حاجن يف اًدج مهم ريثأت وأ رود اهل ةسرامملا ه ذه .اهملعت يتلا ةفرعملا قيبط لأ هن إذ مل ا تتسف ، يمويلا مالكلا يف ةغللا ةسرامم م ريثأت ىلإ رثؤت س.يبل 14 تن لكاشم نم ةلكشملا هذه أش في.اهذيفنتو ةيوغللا ةئيبلاب مامتهالا متي ال ثيح ، ةيوغللا ةئيبلا من دجو ، ثحبلا ةرتف لالخ ال نم برقي ام ثحاب75 ٪ ةطسوتملا ةسردم نم لك نم ةيلخادلا ملسم مامإلا دهعم بالط نم وا.ًاليلق الإ ةيبرعلا ةغللاب نوثدحتي ال ايلعلا ةسردمل وي ةيبالطلا تامظنملا بايغ ىلإ اًضيأ كلذ عجر ال نوموقيو دهعملا اذه يف نوشيعي نيذلا بالطلا ددع ةلقو ، ةغللا لكاشم نع رشابم لكشب ةلوؤسم ب.مهبالطل ةيبرعلا ةغللا ريوطت يف لاعف رود حيث ، ءاقدصألا وأ نيسردملا ءاوس ، مهلوح نم صاخشألا عم اًريثك نولصاوتي بالطلا لازي ال ثدحتي بلل ةيبرعلا ةغللاب ثدحتلل تاريذحتلاو عفادلا اوقلت مهنأ نم مغرلا ىلع ، ةيسينودنإلا ةغ د.اًمئا قلم ةما ةسردملا ةئيب يف ةيبرعلا ةغللا ةسر ؛ هذ وأ ذيفنتل صخشلا ةلواحم يه ةسرامملا ه ت.ةيبرعلا ةغللا ملعت حاجن يف اًدج مهم ريثأت وأ رود اهل ةسرامملا ه ذه .اهملعت يتلا ةفرعملا قيبط لأ هن إذ مل ا تتسف ، يمويلا مالكلا يف ةغللا ةسرامم م ريثأت ىلإ رثؤت س.يبل 14 تن لكاشم نم ةلكشملا هذه أش في.اهذيفنتو ةيوغللا ةئيبلاب مامتهالا متي ال ثيح ، ةيوغللا ةئيبلا من دجو ، ثحبلا ةرتف لالخ ال نم برقي ام ثحاب75 ٪ ةطسوتملا ةسردم نم لك نم ةيلخادلا ملسم مامإلا دهعم بالط نم وا.ًاليلق الإ ةيبرعلا ةغللاب نوثدحتي ال ايلعلا ةسردمل وي ةيبالطلا تامظنملا بايغ ىلإ اًضيأ كلذ عجر ال نوموقيو دهعملا اذه يف نوشيعي نيذلا بالطلا ددع ةلقو ، ةغللا لكاشم نع رشابم لكشب ةلوؤسم ب.مهبالطل ةيبرعلا ةغللا ريوطت يف لاعف رود 595–604. وا ةيبرعلا باتك اوسرد نأ مهل قبس ثيح ةيبرعلا ةغللا يف ةيفلخ مهيدل نيملعملا ءالؤه نأ وه ببسل ب.باتكلا نم ةدام سيردت يف اًكبترم ناك هنأ اوفرتعا يذلاو ، كيدي ني وم نولذبي اولاز ام ، كلذ ع قص.مهبالطل لضفألا ميدقتل مهدهج ىرا أن ؛يه مالكلا قيبطت يف بالطلا دنع فوعض ببسي نيذلا بابسأ ةلق وج دو ملا يبرعلا ملع، عد ةئيبلا م ةيوغللا، ةمظنملا ءاغلإ دعب ةيبرعلا ةغللا قيبطتل ةبقارملا مدع، ةغللا مسق ةصاخ ةيبالطلا، قل طاشن ة تادرفملا ظفح لثم لصفلا جراخ يوغللا، ة لق ةيوغللا ةطشنألا، تغ جهنملا رييتع ميل ةيبرعلا ةغللا، ق ةل ةبغر ال بالط ا ةسارد يف ةيبرعلا ةغلل، ف ةيبرعلا ةغللا ةسرمملا ةلقي ةسردملا ةئيبلا، الوأ احارتقاوي يغبن لمد ,ةيبرعلا ةغللا ىوس ىرخأ ةغللاب بالطلا ىلإ ثدحتلا مدعب ةلواحم ةذتاسألا لكل صصخي نأ دهعملا ري ثم ي ىتح ةيبرعلا ةغللاب داوملا حرش ةلواحمو مألا ةغلب عوضوملا حرش مدع نيملعملل مهملا نم دوعت ال بالط عرعلا ةغللا عامس ىل ةيب ت وشةغللا فلاخملل ةصاخ ةيوغللا ةمظنألا ديد. Aflisia, Noza, and Partomuan Harahap. “Eksisten Bi’ah Lughawiyah Sebagai Media Berbahasa Arab Dalam Meningkatkan Kemampuan Muhadatsah Mahasiswa Prodi Pendidikan Bahasa Arab IAIN Curup.” Lisanul’Arab: Journal of Arabic Learning and Teaching 8, no. 1 (2019): 40– Aflisia, Noza, and Partomuan Harahap. “Eksisten Bi’ah Lughawiyah Sebagai Media Berbahasa Arab Dalam Meningkatkan Kemampuan Muhadatsah Mahasiswa Prodi Pendidikan Bahasa Aflisia, Noza, and Partomuan Harahap. “Eksisten Bi’ah Lughawiyah Sebagai Media Berbahasa Arab Dalam Meningkatkan Kemampuan Muhadatsah Mahasiswa Prodi Pendidikan Bahasa Arab IAIN Curup.” Lisanul’Arab: Journal of Arabic Learning and Teaching 8, no. 1 (2019): 40– 55. Arab IAIN Curup.” Lisanul’Arab: Journal of Arabic Learning and Teaching 8, no. 1 (2019): 40– 55 Arab IAIN Curup.” Lisanul’Arab: Journal of Arabic Learning and Teaching 8, no. 1 (2019) Alfian, Muhammad Evan. “Keterampilan Berbicara Dan Pengajarannya.” Arabia,Jurnal Pendidikan Bahasa Arab 5, no. 1 (2014): 103–21. Alfian, Muhammad Evan. “Keterampilan Berbicara Dan Pengajarannya.” Arabia,Jurnal Pendidikan Bahasa Arab 5, no. 1 (2014): 103–21. Benarioua, Amel. “ةيما لعالا ةغ للا ة سراممو ةيبرعلا ةغللا ن يب مال عالا بلاط.” Human Sciences Journal, October 3, 2021, 595–604. Benarioua, Amel. “ةيما لعالا ةغ للا ة سراممو ةيبرعلا ةغللا ن يب مال عالا بلاط.” Human Sciences Journal, October 3, 2021, 595–604. Fauji, Vina Anisatul, Darul Qutni, and Muchlisin Nawawi. “EFEKTIVITAS MEDIA FLASHCARD TERHADAP KEMAMPUAN MEMBACA NYARING (AL-JAHRIYAH) DAN MEMBACA DALAM HATI (ASH-SHAMITAH) BAHASA ARAB SISWA KELAS VIII MTs NEGERI 1 PURBALINGGA TAHUN AJARAN 2018/2019.” Lisanul Arab: Journal of Arabic Learning and Teaching 9, no. 1 (June 29, 2020): 74–79. https://doi.org/10.15294/la.v9i1.39311. VIII MTs NEGERI 1 PURBALINGGA TAHUN AJARAN 2018/2019.” Lisanul Arab: Journal of Arabic Learning and Teaching 9, no. 1 (June 29, 2020): 74–79. https://doi.org/10.15294/la.v9i1.39311. Fisabilillah, Ibnu, Tiy Kusmarrabbi Karo, and Ilham Tumanggor. AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|41 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|41 AL-HIMAM: Jurnal Ilmu-Ilmu Pendidikan & Bahasa Arab ♦ Volume III, No. 1, Juli-Desember 2023 \|42 وا ةيبرعلا باتك اوسرد نأ مهل قبس ثيح ةيبرعلا ةغللا يف ةيفلخ مهيدل نيملعملا ءالؤه نأ وه ببسل ب.باتكلا نم ةدام سيردت يف اًكبترم ناك هنأ اوفرتعا يذلاو ، كيدي ني وم نولذبي اولاز ام ، كلذ ع قص.مهبالطل لضفألا ميدقتل مهدهج ىرا “Isti’ab al-Mufrodat Wa ’Alaqotuha Binatijah at-Thallab Fi Hifdzi al-Quran.” Al-Himam: Jurnal Ilmu Ilmu wan, Acep. Metodologi Pembelajaran Bahasa Arab. Bandung: PT Rosda Karya, 2013. Hidayat, Nandang Sarip. “اهميلع ت ةءارج إو مالكل ا ةراهم.” Al-Manar 2, no. 8 (2018). https://doi.org/10.24014/al-manar.v2i8.4736. Hidayat, Nandang Sarip. “اهميلع ت ةءارج إو مالكل ا ةراهم.” Al-Manar 2, no. 8 (2018). https://doi.org/10.24014/al-manar.v2i8.4736. Ni’mah, Abdul Wahab Rosyidi dan Mamlu’atul. 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Utari, Anggie Sri. “Atsar Istikhdam Wasilah Dam’iyah al Yad Fi Ta’lim Maharoh al Kalam Bil Madrosah al Ibtidaiyah.” Al-Himam: Jurnal Ilmu Ilmu Pendidikan & Bahasa Arab 2, no. 2 (June 28, 2023): 49–64. https://doi.org/10.51590/alhimam.v2i2.450. سب ةعماج ”.اوغورف نولوك ستاو ةيناثلا ةيموكحلا ةيمالسإلا ةيوناثلا ةسردملا يف مالكلا ةراهم ميلعت تالكشم“ .رهاظ ,اويك ال ,ةيمالسإلا ةنس2013 . سبسا ةعماج ”.اوغورف نولوك ستاو ةيناثلا ةيموكحلا ةيمالسإلا ةيوناثلا ةسردملا يف مالكلا ةراهم ميلعت تالكشم“ .رهاظ ,اويك ال ,ةيمالسإلا ةنس2013 . سر يرصعلا دهعمب يناثلا فصلا بالط ىدل ةيوناثلا ةلحرملا يف مالكلا ةراهم ميلعت تالكشم ليلجت .قدصلا يبسح ,راغي ال ,جنادرس يليد .غنلاغ ةيرمق2022 . ال ,جنادرس يليد .غنلاغ ةيرمق2022 . وا ةيبرعلا باتك اوسرد نأ مهل قبس ثيح ةيبرعلا ةغللا يف ةيفلخ مهيدل نيملعملا ءالؤه نأ وه ببسل ب.باتكلا نم ةدام سيردت يف اًكبترم ناك هنأ اوفرتعا يذلاو ، كيدي ني وم نولذبي اولاز ام ، كلذ ع قص.مهبالطل لضفألا ميدقتل مهدهج ىرا نا ةراهم فعض بابسأ“ .ايلوج سنه ,نويتسال دهعملاب ةطسوتملا ةلحرم يف يناثلا فصلا بالط ىدل ةيبرعلا ةغللاب مالك ال ,ةيمالسإلا ةنسلا ةعماج ”.يوونلا مامإ2021 . نا ةراهم فعض بابسأ“ .ايلوج سنه ,نويتسال دهعملاب ةطسوتملا ةلحرم يف يناثلا فصلا بالط ىدل ةيبرعلا ةغللاب مالك ال ,ةيمالسإلا ةنسلا ةعماج ”.يوونلا مامإ2021 . ال ,ةيمالسإلا ةنسلا ةعماج ”.يوونلا مامإ2021 .
https://openalex.org/W3114972301	http://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0040-1714133.pdf	English	null	Reproductive Outcomes in Cases of Subclinical Hypothyroidism and Thyroid Autoimmunity: A Narrative Review	Revista brasileira de ginecologia e obstetrícia	2,020	cc-by	5,094	Keywords Abstract Thyroid diseases are relatively common in women in the reproductive period. It is currently understood that clinically-evident thyroid disorders may impair ovulation and, consequently, fertility. However, to date it has not been proven that high serum levels of thyroid-stimulating hormone and/or positivity for antithyroid antibodies are associated to a reduction in fertility, mainly in the absence of altered thyroxine levels. The present comprehensive review aims to present current data on the association between subclinical hypothyroidism and/or thyroid autoimmunity and reproductive outcomes. ►thyroid diseases ►thyroid function tests ►thyroid hormones ►hypothyroidism ►autoimmunity ►female infertility Article published online: 2020-12-21 Article published online: 2020-12-21 Article published online: 2020-12-21 Review Article 829 Review Article 829 Resultados reprodutivos nos casos de hipotireoidismo subclínico e autoimunidade tireoidiana: Uma revisão narrativa Address for correspondence Bruno Ramalho de Carvalho, MD, MSc, SGAS 614, Conjunto C, Sala 177, Edifício Vitrium, Centro Médico Inteligente, Asa Sul, Brasília, Distrito Federal, 70200-740, Brazil (e-mail: ramalho.b@gmail.com). 1Hospital Sírio-Libanês, Brasília, Distrito Federal, Brazil 3Department of Obstetrics and Gynecology, School of Medical Sciences, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil p p 4Department of Gynecology and Obstetrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil 5Discipline of Gynecology, Department of Obstetrics and Gynecology, Hospital das Clínicas - HCFMUSP, Faculdade de Medina da Universidade de São Paulo, SP, Brazil Rev Bras Ginecol Obstet 2020;42(12):829–833. Rev Bras Ginecol Obstet 2020;42(12):829–833. Keywords ►thyroid diseases ►thyroid function tests ►thyroid hormones ►hypothyroidism ►autoimmunity ►female infertility Abstract Palavras-chave ►doenças da tireoide ►testes de função da tireoide ►hormônios da tireoide ►hipotireoidismo ►autoimunidade ►infertilidade feminina Resumo received May 19, 2020 accepted June 3, 2020 1Hospital Sírio-Libanês, Brasília, Distrito Federal, Brazil 2Human Reproduction Unit, Hospital Fêmina, Grupo Hospitalar Conceição, Porto Alegre, Rio Grande do Sul, Brazil 3Department of Obstetrics and Gynecology, School of Medical Sciences, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil 4Department of Gynecology and Obstetrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil 5Discipline of Gynecology, Department of Obstetrics and Gynecology, Hospital das Clínicas - HCFMUSP, Faculdade de Medina da Universidade de São Paulo, SP, Brazil Resumo As doenças da tireoide são relativamente comuns em mulheres no período reprodu- tivo. Atualmente, entende-se que distúrbios da tireoide clinicamente evidentes podem prejudicar a ovulação e, consequentemente, a fertilidade. No entanto, não se provou até o presente que níveis séricos altos do hormônio estimulador da tireoide e/ou positividade para anticorpos antitireoidianos estão associados a uma redução na fertilidade, sobretudo na ausência de níveis alterados de tiroxina. Esta revisão narrativa tem como objetivo apresentar dados atuais sobre a associação entre hipotireoidismo subclínico e/ou autoimunidade tireoidiana e resultados reprodutivos. Palavras-chave ►doenças da tireoide ►testes de função da tireoide ►hormônios da tireoide ►hipotireoidismo ►autoimunidade ►infertilidade feminina Address for correspondence Bruno Ramalho de Carvalho, MD, MSc, SGAS 614, Conjunto C, Sala 177, Edifício Vitrium, Centro Médico Inteligente, Asa Sul, Brasília, Distrito Federal, 70200-740, Brazil (e-mail: ramalho.b@gmail.com). DOI https://doi.org/ 10.1055/s-0040-1714133. ISSN 0100-7203. Rev Bras Ginecol Obstet 2020;42(12):829–833. Subclinical Hypothyroidism Subclinical hypothyroidism is a condition in which the level of TSH is elevated, but the level of fT4 is normal. It represents an early, mild thyroid failure, and affects up to 10% of the adult population.27 However, its clinical signiﬁcance has not been consistently proven. Raber et al7 followed 223 women for upto 5 years, and they observed lower conception rates among women who never achieved a basal TSH < 20 mUI/L with fT4 therapy, and, then, they suggested a negative effect of such ﬁndings on reproductive function (level 4). Moreover, the meta-analysis of two studies21 showed a signiﬁcant decrease in the rates of miscarriage (relative risk [RR]: 0.18; 95%; 95% conﬁdence interval [95%CI]: 0.08–0.39; p < 0.01) and preterm delivery (RR: 0.41; 95%CI: 0.24–0.68; p ¼ 0.0005) in women with SCH treated with levothyroxine (LT4) (level 1). However, contrary to those authors, preconceptionTSH 2.5 mIU/L was not associated to time to biochemical pregnancy (odds ratio [OR]:1.09;95%CI: 0.90–1.31), pregnancyloss (RR: 1.15; 95%CI: 0.86–1.54) or live births (RR: 1.01, 95%CI: 0.89–1.14) among 1,193 women with normal fT4 (0.7 ng/dL to 1.85 ng/dL) and a history of either one or two previous pregnancy losses, even if they were positive for anti-thyroid antibodies (TGAb 115 - IU/mL and/or TPOAb 35 IU/mL), and after adjusting for age and body massindex. Theauthorsalso attempted to determine a TSH cut-off affecting the continuation of pregnancy, but an additional analysis ofboth TSH tertiles andcontinuousTSHdid not result in differences between women with TSH 2.5 mIU/L and those with TSH < 2.5 mIU/L (level 2).12 There are some studies evaluating the effect of LT4 in women with subclinical hypothyrodism undergoing assisted reproductive treatments, like in vitro fertilization (IVF), with conﬂicting results.8,10,15,22,26 According to Kim et al,10 women with subclinical hypothyroidism undergoing assisted reproductive techniques presented with similar clinical pregnancy rates when compared with controls, despite the signiﬁcant differ- ences in the number of good-quality embryos, implantation rates, and live-birth rates (RR: 1.8; 95%CI: 1.0–3.25; p ¼ 0.05; and RR: 2.13; 95%CI: 1.07–4.21; p ¼ 0.03 respectively).10 How- ever, the miscarriage rate was signiﬁcantly lower in the LT4 group (no miscarriages versus 33,3% in the control group; p ¼ 0.021) (level 2). In another trial,8 LT4 or placebo were initiated one month before IVF and were maintained through- out pregnancy. Introduction selected for this review.7–19 The references of the selected studies were also checked, and seven more relevant articles were included.20–26Theevidencewashierarchizedaccording to the Oxford Centre for Evidence-based Medicine’s 2011 Levels of Evidence6 by the ﬁrst author (BRC), and checked by the second author (APN); there were no discordances between them or between them and the other authors. The thyroid gland is responsible for regulating several mech- anisms of human physiology, which include the reproductive function. Thyroid hormones are involved in the modulation of the hypothalamic-pituitary-gonadal axis and, despite the lack of consistent scientiﬁc evidence, it is currently understood that clinically evident thyroid disorders may impair ovulation and, consequently, fertility.1 Thyroid diseases arerelativelycommon in women in the reproductive period. A signiﬁcant association between clinical thyroid disorders and abnormalities of the reproductive system has been largely conﬁrmed: both primary hyperthyroidism and hypothyroidism have been documented to produce variable degrees of gonadal dysfunction. Neverthe- less, the impact of subclinical thyroid dysfunction and/or thyroid autoimmunity (TAI) on fertility and reproductive out- comes is not consensual, although they may be related to infertility and the risk of spontaneous pregnancy loss.1,2 As a matter of course, subclinical hypothyroidism (SCH) has been deﬁned as a level of thyroid stimulating hormone (TSH) going over theupper threshold of4.5 mIU/Lto5.0mIU/Linthesetting ofa normal level of freethyroxine(fT4).3 Nonetheless,thelimits commonly vary among studies, and it has been suggested that the upper cutoff forTSH shouldbeset at 2.5 mIU/L, basedonthe observation that 95% of asymptomatic people have that level or even lower levels of TSH.4 Regarding thyroid diseases, in addition to idiopathic changes in function, situations resulting from the presence of autoantibodies are quite common, such as Hashimoto thyroiditis and Graves disease. Currently, at least three anti-thyroid antibodies can be evaluated in human serum: the thyroid globulin antibody (TGAb), the thyroid peroxidase antibody (TPOAb), and the thyrotropin receptor antibody (TRAb). However, presenting anti-thyroid antibodies is not sufﬁcient to develop autoimmune thyroid disease, which pathophysiology is not yet fully understood. Thus, the clinical relevance of presenting positive antibodies without an estab- lished disease is still questionable, including the inﬂuence on fertility.5 The present narrative review aims to present the current data on the association between SCH and/or TAI and reproductive outcomes. Our objective is to help clinicians decide the medical approach to women attempting to conceive and presenting those conditions, based on hierarchized evi- dence. Palavras-chave ►doenças da tireoide ►testes de função da tireoide ►infertilidade feminina Copyright © 2020 by Thieme Revinter Publicações Ltda, Rio de Janeiro, Brazil DOI https://doi.org/ 10.1055/s-0040-1714133. ISSN 0100-7203. received May 19, 2020 accepted June 3, 2020 Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. 830 Subclinical Hypothyroidism The number of follicles punctured, mature oocytes, and the fertilization, pregnancy, and delivery rates were signiﬁcantly higher in the treatment group. Moreover, the miscarriageratewassigniﬁcantlylower intheintervention group (level 1).8 In a cohort study by Cai et al,15 270 women with SCH supplemented with LT4 before IVF were compared with 200 age-matched euthyroid women who underwent classical IVF or intracytoplasmic sperm injection (ICSI) In Introduction However, once the levels of evidence do not provide a deﬁnitive judgment about the quality of the studies included nor they constitute a ﬁnal recommendation,6 clinicians may apply individualization as the main key for their critical appraisal in treatment decisions. Rev Bras Ginecol Obstet Vol. 42 No. 12/2020 Methods The authors did not ﬁnd a signiﬁcant delay in pregnancy (OR: 1.11; 95%CI: 0.88–1.40), higher risk of pregnancy loss (RR: 0.90; 95%CI: 0.61–1.33) or impaired live birth rate in women with circu- lating anti-thyroid antibodies (RR: 1.04; 95%CI: 0.90–1.20), even after adjusting for age and body mass index (level 2).12 Addiotnally, according to van den Boogaard et al,9,29 no association was found between TAI and the rates of clinical pregnancy after IVF in the meta-analysis of seven studies (OR: 0.71;95%CI:0.36–1.4).However,thesamestudyfoundelevated odds for unexplained subfertility (OR: 1.47; 95%CI: 1.06–2.02; p ¼ 0.02), miscarriage (OR: 3.73; 95%CI: 1.83–7.6; p ¼ 0.0003), recurrent miscarriage(OR:2.26;95%CI: 1.46–73.5; p ¼ 0.0003), and preterm delivery (OR: 1.93; 95%CI: 1.08–3.47; p ¼ 0.03) among euthyroid women positive for thyroid autoantibodies (level 1).9,29 In the same sense, the meta-analysis20 of seven homoge- neous cohort studies demonstrated a signiﬁcant elevation in the odds of miscarriage among subfertile women presenting with thyroid autoantibodies (OR: 3.15; 95%CI: 2.23–4.44; p < 0.001), especially TPOAb, but such an association was not proven byanalyzing the three eligiblestudies involving women with recurrent pregnancy loss. Moreover, the authors found a 2-fold increase in the odds of preterm birth in the presence of TAI (OR: 2.07; 95%CI: 1.17–3.68; p ¼ 0.01), with a signiﬁcant 52% reduction in the relative risk of miscarriage (RR: 0.48; 95% CI: 0.25- 0.92; p ¼ 0.03) and a 69% reduction in the relative risk of preterm birth (RR: 0.31 95%CI: 0.11–0.9; p < 0.05) when LT4 was supplemented in women with thyroid autoantibodies (level 1).20 Finally, in a recent meta-analysis, Dong et al19 showed an association between TAI and recurrent pregnancy loss (OR: 1.94; 95%CI: 1.43–2,.4), but LT4 did not improve the pregnancy outcomes (level 1).19 Finally, according to the American Society for Reproductive Medicine,3 evidence that SCH (deﬁned as TSH > 2.5 mIU/L with a normal level of fT4) affects fertility or induces miscar- riages is insufﬁcient. In the absence of speciﬁc recommenda- tions for women attempting pregnancy, there is a suggestion to use pregnancy thresholds to minimize the potential risks associated with SCH. The American Thyroid Association2 has published recommendations on the thresholds; brieﬂy, in the absence of TAI, LT4 replacement is recommended for women presenting with TSH > 10.0 mIU/L (strong recommendation, but based on low-quality evidence), and could be considered for those presenting with TSH 4.0 mIU/L and < 10.0 mIU/L (weak recommendation, also based on low-quality evidence). Methods Only in one study involving 64 women with both subclinical hypothyroidism and positive or negative TPOAb, LT4 replacement provided an improvement in the rate of live births (RR: 2.13; 95%CI: 1.07–4.21), with similar miscarriage rates (RR: 0.11; 95%CI: 0.01–1.98) (level 1).18 Nevertheless, the authors could not drawclearconclusions dueto thelow tovery low quality of the evidence reported. Finally according to the American Society for Reproductive subfertility and adverse pregnancy outcomes, like miscarriage, recurrent miscarriage and preterm birth. Although not consen- sual, the literature suggests that the administration of LT4 can improvereproductiveoutcomesinwomenwithnormalthyroid function and positive thyroid autoantibodies (level 1).9,20,28 total, 176 out of 270 women completed all pregnancy visits of the study, and were included in the ﬁnal analysis. In the SCH treated with LT4 and euthyroid groups of women who under- went IVF in the same period, the overall rates of clinical pregnancy (44.31% versus 38.36%; p ¼ 0.251 respectively) and miscarriage (10.3% versus 10.7%, p ¼ 0.39 respectively) were similar (level 3).15 Moreover, another study12 demon- strated that the treatment with LT4 lead to the same rates of clinical pregnancy, miscarriage, and live births, which were independent of TSH levels, in women with SCH. There were no differences between the groups regarding the total number of oocytes retrieved and good-quality embryos (level 2).12 To enhance the challenge, two recent meta-analysis have shown thatLT4supplementationinwomenwithSCHcansigniﬁcantly reduce the risk of miscarriage after assisted reproductive technologies (ARTs) in almost 50% (RR: 0.51; 95%CI: 0.32–0.82), but not the rate of preterm birth (RR: 1.13; 95% CI: 0.65–1.96). In women with TAI, LT4 supplementation reduced the risks of pregnancy loss (RR: 0.61; 95%CI: 0.39–0.96; p ¼ 0.03) and preterm birth (RR: 0.49; 95%CI: 0.30–0.79; p ¼ 0.003) in naturally-conceived pregnancies, but not in pregnancies achieved by ARTs (level 1).17 These results support previous similarly-designed studies (level 1).16,22 As a matter of fact, the last Cochrane review23 con- cluded that evidence is not sufﬁcient to support the recom- mendation of one prepregnancy or mid-pregnancy intervention over another, in cases of SCH (level 1). Given that their ﬁndings were only based on two trials with a moderate risk of bias, and that new trials have been published after that, the conclusion of a reduction in preterm birth and a trend toward reduced miscarriage with the use of LT4 therapy should be taken with caution when deciding to treat euthyroid women. Methods Despite the aforementioned ﬁndings, Vissenberg et al21 could not demonstrate the beneﬁts of treating euthyroid wom- enwith positive thyroid autoantibodies with LT4 (level 1).21In a preview cohort study, Raber et al7 did not ﬁnd a signiﬁcant association between the presence of TPOAb and TGAb and pregnancy or abortion rates in infertile women with or without SCHfollowed-upfor morethan5years(level4).Inaccordanceto those results, Dhillon-Smith et al25 could not ﬁnd signiﬁcant differences in the rates of live births after at least 34 weeks of pregnancy by using 50 µg of LT4 once a day, started before conception and continued throughout pregnancy, among eu- thyroid women with TPOAb with a history of miscarriage or infertility. There was also no signiﬁcant effect of LT4 on other pregnancy or neonatal outcomes, including the incidence of miscarriage and preterm birth (level 1).25 Methods More recently, a Cochrane systematic review18 evalu- ated the LT4 treatment in subfertile women with SCH under- going ARTs. Only in one study involving 64 women with both subclinical hypothyroidism and positive or negative TPOAb, LT4 replacement provided an improvement in the rate of live births (RR: 2.13; 95%CI: 1.07–4.21), with similar miscarriage rates (RR: 0.11; 95%CI: 0.01–1.98) (level 1).18 Nevertheless, the authors could not drawclearconclusions dueto thelow tovery low quality of the evidence reported. First of all, the association between TAI and impaired fertility is still to be proven. In the study by Plowden et al,12 TAI (TGAb 115 IU/mL and/or TPOAb 35 IU/mL) was ex- amined in relation to time to biochemical pregnancy, preg- nancy loss, and live birth among women with normal fT4 with one or two previous pregnancy losses. The authors did not ﬁnd a signiﬁcant delay in pregnancy (OR: 1.11; 95%CI: 0.88–1.40), higher risk of pregnancy loss (RR: 0.90; 95%CI: 0.61–1.33) or impaired live birth rate in women with circu- lating anti-thyroid antibodies (RR: 1.04; 95%CI: 0.90–1.20), even after adjusting for age and body mass index (level 2).12 Addiotnally, according to van den Boogaard et al,9,29 no association was found between TAI and the rates of clinical pregnancy after IVF in the meta-analysis of seven studies (OR: 0.71;95%CI:0.36–1.4).However,thesamestudyfoundelevated odds for unexplained subfertility (OR: 1.47; 95%CI: 1.06–2.02; p ¼ 0.02), miscarriage (OR: 3.73; 95%CI: 1.83–7.6; p ¼ 0.0003), recurrent miscarriage(OR:2.26;95%CI: 1.46–73.5; p ¼ 0.0003), and preterm delivery (OR: 1.93; 95%CI: 1.08–3.47; p ¼ 0.03) among euthyroid women positive for thyroid autoantibodies (level 1).9,29 First of all, the association between TAI and impaired fertility is still to be proven. In the study by Plowden et al,12 TAI (TGAb 115 IU/mL and/or TPOAb 35 IU/mL) was ex- amined in relation to time to biochemical pregnancy, preg- nancy loss, and live birth among women with normal fT4 with one or two previous pregnancy losses. Methods Using the keywords subclinical hypothyroidism, thyroid autoim- munity, and infertility, we searched for clinical trials, controlled clinical trials, meta-analyses, and randomized controlled trials on the following databases: PubMed, the Cochrane Database of SystematicReviews,theCochraneCentralRegisterofControlled Trials, the Cochrane Gynecology and Fertility Group specialized register, and clinicaltrials.gov. No date or language restrictions were applied to the search. A total of 13 studies were primarily Rev Bras Ginecol Obstet Vol. 42 No. 12/2020 Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. 831 total, 176 out of 270 women completed all pregnancy visits of the study, and were included in the ﬁnal analysis. In the SCH treated with LT4 and euthyroid groups of women who under- went IVF in the same period, the overall rates of clinical pregnancy (44.31% versus 38.36%; p ¼ 0.251 respectively) and miscarriage (10.3% versus 10.7%, p ¼ 0.39 respectively) were similar (level 3).15 Moreover, another study12 demon- strated that the treatment with LT4 lead to the same rates of clinical pregnancy, miscarriage, and live births, which were independent of TSH levels, in women with SCH. There were no differences between the groups regarding the total number of oocytes retrieved and good-quality embryos (level 2).12 To enhance the challenge, two recent meta-analysis have shown thatLT4supplementationinwomenwithSCHcansigniﬁcantly reduce the risk of miscarriage after assisted reproductive technologies (ARTs) in almost 50% (RR: 0.51; 95%CI: 0.32–0.82), but not the rate of preterm birth (RR: 1.13; 95% CI: 0.65–1.96). In women with TAI, LT4 supplementation reduced the risks of pregnancy loss (RR: 0.61; 95%CI: 0.39–0.96; p ¼ 0.03) and preterm birth (RR: 0.49; 95%CI: 0.30–0.79; p ¼ 0.003) in naturally-conceived pregnancies, but not in pregnancies achieved by ARTs (level 1).17 These results support previous similarly-designed studies (level 1).16,22 As a matter of fact, the last Cochrane review23 con- cluded that evidence is not sufﬁcient to support the recom- mendation of one prepregnancy or mid-pregnancy intervention over another, in cases of SCH (level 1). Given that their ﬁndings were only based on two trials with a moderate risk of bias, and that new trials have been published after that, the conclusion of a reduction in preterm birth and a trend toward reduced miscarriage with the use of LT4 therapy should be taken with caution when deciding to treat euthyroid women. More recently, a Cochrane systematic review18 evalu- ated the LT4 treatment in subfertile women with SCH under- going ARTs. Final Considerations In the absence of speciﬁc recommendations for women attempting pregnancy, some defend the use of pregnancy thresholds to minimize the potential risks associated with TAI. According to the most recent American Thyroid Associa- tion (ATA) recommendations,2 pregnant women presenting withTSH > 2.5 mIU/L shouldberegularlyevaluatedfor TPOAb; brieﬂy, for those TPOAb-positive,LT4 therapy is recommended if TSH 4.0 mIU/L (strong recommendation, based on moder- ate-quality evidence), and could be considered for those presenting with TSH > 2.5 mIU/L and < 4.0 mIU/L (weak rec- ommendation, also based on moderate-quality evidence). The decision to treat SCH, particularly in women attempting pregnancy and infertile women, remains controversial, since the current understanding of the effect of thyroid dysfunction and/or autoimmunity on reproductive outcomes is based largely on low quality evidence. For this reason, the ﬁndings on the reproductive inﬂuence of SCH and TAI should be considered with care. Also because of the lacking evidence, the treatment with LT4 should not be established as a routine for women with SCH or those positive for thyroid autoanti- bodies as isolated ﬁndings, even assuming that potential beneﬁts may outweigh the potential risks. As a matter of fact, the use LT4 will certainly beneﬁt pregnant women with clinical hypothyroidism, and is an accepted strategy for those with a combination of TAI and elevated TSH. The treatment may be extended with caution and informed consent when that combination is found in subfertile women or those attempting pregnancy, but future better-designed studies are expected to support strong recommendations. Finally, screening for thyroid dysfunctions may be considered reason- able in women who are attempting to conceive and in the initial stage of pregnancy, but this is not consensual. Regarding the treatment with LT4, it is well established only in cases of clinical hypothyroidism, but it should be accepted in the following situations: 1) SCH associated to infertility; 2) TAI withTSH 4.0mIU/L;or3)whenTSH > 10.0mIU/L.Therefore, it should not be a rule for subclinical conditions, especially in the absence of autoimmunity. In the case of an individualized treatment for those who are candidates for maternity, we suggest that the same guidelines provided for pregnancy should be followed. Laboratorial Pitfalls Thyroid function tests (TFTs) are routinely ordered, but the evaluation and interpretation of the results may be difﬁcult at times due to technical problems. The pitfalls in the hormonal evaluation can be preanalytical, analytical, and postanalytical. The preanalytical factors include age, preg- nancy, use of medications (such as oral contraceptives and biotin), genetic mutations, systemic diseases, and critical illnesses. The analytical errors occur due to heterophile antibodies and macro-TSH. The postanalytical errors include wrong registration of the result by the laboratory, mistakes in the units of the parameter checked, and failure to identify the normal data.28,30 Thus, before taking clinical decisions, it is important that the physician become aware of those challenges and repeat the test in case of doubt. Beside all the lack of evidence to support the use of LT4 in euthyroid women with positive antibodies, Bartáková et al11 analyzed the reproductive outcomes of 258 women up to 47 months after an episode of spontaneous abortion in the ﬁrst trimester, and 43% of themwere “positivefor thyroid disorders” (level 3). Despite the fact that they found a signiﬁcantly lower rate of secondary infertility among women treated with LT4 when compared with the controls and untreated women (4.1% versus 10.9% versus 21.1% respectively), such a ﬁnding was not clear whentheycomparedthecontrolstotreatedand untreated positive women together (10.9% versus 9.9% respectively). The authors concluded that screening for thyroid disorders in women after spontaneous abortion and treatment with LT4 is cost-saving and improves the subsequent pregnancy rate. Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. Another trial14 evaluated the treatment with LT4 initiated between 2 and 4 weeks before the controlled ovarian hyper- stimulation for IVFand continued through the end of pregnancy in women with normal thyroid function who tested positive for TPOAb. The LT4 treatment did not reduce rates of miscarriage or improved the rates of live births compared with the usual care (level 1).14 A recent Cochrane Systematic Review18 also showed no differences in miscarriage rates or live-birth rates with the LT4 treatment or placebo in a similar group of women under- going ARTs (level 1). 4. The LT4 therapy is not recommended (strong reco dation, evidence of high quality) for women prese (a) TSH 2.5 mIU/mL; (b) TPOAb-negative, TSH < 4.0 mIU/L.2 4. The LT4 therapy is not recommended (strong recommen- dation, evidence of high quality) for women presenting: (a) TSH 2.5 mIU/mL; (b) TPOAb-negative, TSH < 4.0 mIU/L.2 Thyroid Autoimmunity Thyroid autoimmunity seems to be relatively common among women of reproductive age, and it might be associated with Rev Bras Ginecol Obstet Vol. 42 No. 12/2020 832 References 1 Gracia CR. Thyroid in Reproduction. Semin Reprod Med. 2016;34 (06):315–316. Doi: 10.1055/s-0036-1593490 16 Li J, Shen J, Qin L. Effects of levothyroxine on pregnancy outcomes in women with thyroid dysfunction: a meta-analysis of randomized controlled trials. Altern Ther Health Med. 2017;23(02):49–58 2 Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, et al. 2017 Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the postpartum. Thyroid. 2017;27(03):315–389. Doi: 10.1089/thy.2016.0457 17 Rao M, Zeng Z, Zhao S, Tang L. Effect of levothyroxine supplemen- tation on pregnancy outcomes in women with subclinical hypo- thyroidism and thyroid autoimmuneity undergoing in vitro fertilization/intracytoplasmic sperm injection: an updated meta-analysis of randomized controlled trials. Reprod Biol Endo- crinol. 2018;16(01):92. Doi: 10.1186/s12958-018-0410-6 3 Practice Committee of the American Society for Reproductive Medicine. Subclinical hypothyroidism in the infertile female population: a guideline. Fertil Steril. 2015;104(03):545–553. 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Thyrox- ine treatment modiﬁed in infertile women according to thyrox- ine-releasing hormone testing: 5 year follow-up of 283 women referred after exclusion of absolute causes of infertility. Hum Reprod. 2003;18(04):707–714. References Doi: 10.1093/humrep/deg142 22 Velkeniers B, Van Meerhaeghe A, Poppe K, Unuane D, Tournaye H, Haentjens P. Levothyroxine treatment and pregnancy outcome in women with subclinical hypothyroidism undergoing assisted reproduction technologies: systematic review and meta-analysis of RCTs. Hum Reprod Update. 2013;19(03):251–258. Doi: 10.1093/humupd/dms052 8 Abdel Rahman AH, Aly Abbassy H, Abbassy AAE. Improved in vitro fertilization outcomes after treatment of subclinical hypothyroid- ism in infertile women. Endocr Pract. 2010;16(05):792–797. Doi: 10.4158/EP09365.OR 23 Reid SM, Middleton P, Cossich MC, Crowther CA, Bain E. Inter- ventions for clinical and subclinical hypothyroidism pre-preg- nancy and during pregnancy. Cochrane Database Syst Rev. 2013; (05):CD007752. Doi: 10.1002/14651858.CD007752.pub3 9 van den Boogaard E, Vissenberg R, Land JA, van Wely M, van der Post JA, Goddijn M, et al. Signiﬁcance of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review. Hum Reprod Update. 2011; 17(05):605–619. Doi: 10.1093/humupd/dmr024 24 Negro R, Schwartz A, Stagnaro-Green A. Impact of levothyroxine in miscarriage and preterm delivery rates in ﬁrst trimester thyroid antibody positive women with TSH less than 2.5 mIU/L. J Clin Endocrinol Metab. 2016;101(10):3685–3690. Doi: 10.1210/ jc.2016-1803 10 Kim CH, Ahn JW, Kang SP, Kim SH, Chae HD, Kang BM. Effect of levothyroxine treatment on in vitro fertilization and pregnancy outcome in infertile women with subclinical hypothyroidism under- going in vitro fertilization/intracytoplasmic sperm injection. Fertil Steril.2011;95(05):1650–1654.Doi:10.1016/j.fertnstert.2010.12.004 25 Dhillon-Smith RK, Middleton LJ, Sunner KK, Cheed V, Baker K, Farrell-Carver S, et al. Levothyroxine in women with thyroid peroxidase antibodies before conception. N Engl J Med. 2019; 380(14):1316–1325. Doi: 10.1056/NEJMoa1812537 11 Bartáková J, Potluková E, Rogalewicz V, Fait T, Schöndorfová D, Telička Z, et al. Screening for autoimmune thyroid disorders after spontaneous abortion is cost-saving and it improves the subse- quent pregnancy rate. BMC Pregnancy Childbirth. 2013;13:217. Doi: 10.1186/1471-2393-13-217 26 Rao M, Zeng Z, Zhou F, Wang H, Liu J, Wang R, et al. Effect of levothyroxine supplementation on pregnancy loss and preterm birth in women with subclinical hypothyroidism and thyroid auto- immunity: a systematic review and meta-analysis. Hum Reprod Update. 2019;25(03):344–361. Doi: 10.1093/humupd/dmz003 12 Plowden TC, Schisterman EF, Sjaarda LA, Zarek SM, Perkins NJ, Silver R, et al. Subclinical hypothyroidism and thyroid autoim- munity are not associated with fecundity, pregnancy loss, or live birth. J Clin Endocrinol Metab. 2016;101(06):2358–2365. Doi: 10.1210/jc.2016-1049 27 Biondi B, Cappola AR, Cooper DS. Subclinical hypothyroidism: a review.JAMA.2019;322(02):153–160.Doi:10.1001/jama.2019.9052 28 Garg MK, Mahalle N, Hari Kumar KVS. Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. Reproductive Outcomes in cases of Subclinical Hypothyroidism and Thyroid Autoimmunity Carvalho et al. 833 Reprod Biol Endocrinol. 2017;15(01):39. Doi: 10.1186/s12958- 017-0257-2 Practical Aspects In the absence of sufﬁciently consistent scientiﬁc evidence on the approach of thyroid function in women attempting to conceive, and considering the aforementioned ﬁndings, we propose the following practical aspects: 1. Healthy women actively attempting to conceive should not be evaluated for thyroid disorders (strong recommen- dation, evidence of moderate quality);12 2. Infertile women shouldbe evaluated for thyroid disorders;3 3. In infertile women presenting with TSH > 2.5 mIU/L, evaluate the TPOAb: (a) The LT4 therapy is recommended (strong recommenda- tion, evidence of low to moderate quality) for women presenting: i. TPOAb-positive, TSH 4.0 mIU/L; ii. TPOAb-negative, TSH > 10.0 mIU/L; (b) The LT4 therapy may be individually considered (weak recommendation, evidence of moderate quality) for women presenting: i. TPOAb-positive, TSH > 2.5 mIU/L and < 4.0 mIU/L; 2 Conﬂict of Interests The authors have no conﬂict of interests to declare. i. TPOAb positive, TSH > 2.5 mIU/L and < 4.0 mIU/L; ii. TPOAb-negative, TSH 4.0 mIU/L and < 10.0 mIU/L;2 TPOAb-negative, TSH 4.0 mIU/L and < 10.0 mIU/L;2 Rev Bras Ginecol Obstet Vol. 42 No. 12/2020 References Laboratory evaluation of thyroid function: dilemmas and pitfalls. Med J DY Patil Univ. 2016;9(04):430–436 13 Busnelli A, Paffoni A, Fedele L, Somigliana E. The impact of thyroid autoimmunity on IVF/ICSI outcome: a systematic review and meta-analysis. Hum Reprod Update. 2016;22(06):775–790. Doi: 10.1093/humupd/dmw019 29 van den Boogaard E, Vissenberg R, Land JA, van Wely M, ven der Post JA, Goddijn M, et al. Signiﬁcance of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnan- cy: a systematic review. Hum ReprodUpdate. 2016;22(04):532–533. Doi: 10.1093/humupd/dmw003 14 Wang H, Gao H, Chi H, Zeng L, Xiao W, Wang Y, et al. Effect of levothyroxine on miscarriage among women with normal thyroid function and thyroid autoimmunity undergoing in vitro fertiliza- tion and embryo transfer: a randomized clinical trial. JAMA. 2017; 318(22):2190–2198. Doi: 10.1001/jama.2017.18249 30 Vandendriessche B, Lapauw B, Kaufman JM, Fiers T. A practical approach towards the evaluation of aberrant thyroid function tests. Acta Clin Belg. 2020;75(02):155–162. Doi: 10.1080/17843286.2019.1577531 15 Cai Y, Zhong L, Guan J, Guo RJ, Niu B, Ma YP, et al. Outcome of in vitro fertilization in women with subclinical hypothyroidism. Rev Bras Ginecol Obstet Vol. 42 No. 12/2020
https://openalex.org/W4392869494	https://journal.unnes.ac.id/nju/abdimas/article/download/40015/13808	Indonesian	null	Internalisasi Nilai-Nilai Konservasi Lingkungan Melalui Penuturan Cerita Rakyat Bagi Anak-Anak di Desa Bedono Kecamatan Sayung Kabupaten Demak	Jurnal abdimas/Jurnal Abdimas	2,022	cc-by	3,758	Abdimas Vol 26, No. 2 (2022): December 2022 Abdimas Vol 26, No. 2 (2022): December 2022 ABDIMAS Jurnal Pengabdian kepada Masyarakat https://journal.unnes.ac.id/nju/index.php/abdimas/ Jurnal Pengabdian kepada Masyarakat https://journal.unnes.ac.id/nju/index.php/abdimas/ Abstrak Kerusakan lingkungan wilayah pesisir terjadi tidak hanya karena bencana alam saja, namun disebabkan juga oleh aktivitas manusia. Wilayah Desa Bedono, Kecamatan Sayung, Kabupaten Demak saat ini sudah kehilangan sepertiga wilayahnya karena abrasi laut. Penanaman mangrove sudah dilakukan, namun kesadaran masyarakat untuk memelihara dan melestarikan mangrove masih rendah. Hal itu dikarenakan, kurangnya sosialisasi dalam keluarga terutama kepada anak-anak tentang nilai konservasi lingkungan. Sehingga diperlukan suatu kegiatan bagi anak-anak untuk menginternalisasikan nilai-nilai konservasi lingkungan melalui media yang menarik perhatian anak-anak secara menyenangkan. Berdasarkan alasan tersebut, tim pengabdian dari Fakultas Ilmu Sosial Universitas Negeri Semarang mengajukan kegiatan berupa internalisasi nilai-nilai konservasi lingkungan melalui penuturan cerita rakyat bagi anak-anak di Desa Bedono Kecamatan Sayung Kabupaten Demak. Adapun tahapan kegiatan untuk menyelesaikan permasalahan di Desa Bedono ini yaitu: pemetaan masalah dan potensi melalui FGD, wawancara, dan observasi; pelatihan pembuatan media dan pendampingan; kegiatan dengan para sesepuh; dan simulasi penggunaan media. Dengan kegiatan ini bertujuan untuk dapat menggugah kesadaran anak-anak untuk berperan dalam upaya rehabilitasi mangrove dan menjaga lingkungan. Bertambahnya ilmu pengetahuan bagi anak-anak secara khusus dan masyarakat Desa Bedono secara umum. Ilmu pengetahuan ini berasal dari kearifan lokal masyarakat lokal melalui penuturan pengalaman para sesepuh desa. Kata kunci : : cerita rakyat, kearifan lokal, internalisasi nilai, konservasi lingkungan Internalisasi Nilai-Nilai Konservasi Lingkungan Melalui Penuturan Cerita Rakyat Bagi Anak-Anak di Desa Bedono Kecamatan Sayung Kabupaten Demak Atika Wijaya, Asma Luthfi, Fajar, Harto Wicaksono, Fadly Husain Universitas Negeri Semarang, Indonesia Abdimas Vol 26, No. 2 (2022): December 2022 terendam air laut (Nugroho dkk., 2020; Zaky dkk., 2012; Chafid dkk., 2012). Pemerintah pusat melalui Kemenko Maritim turut serta mengkoordinir upaya penanggulangan bencana abrasi ini (Biro Perencanaan dan Informasi, 2018). Khusus untuk penanaman mangrove, pemerintah daerah bekerja sama dengan OISCA, LSM Internasional Jepang, melakukan konservasi mangrove dengan cara melakukan pembibitan, penanaman, dan pemeliharaan mangrove, dengan pendekatan personal, maupun pendekatan kelembagaan. Sejak tahun 2004, upaya konservasi mangrove giat dilakukan dan menjadi salah satu pilot project program rehabilitasi mangrove yang dilaksanakan oleh OISCA Jepang yang bekerja sama dengan Pemerintah Daerah Kabupaten Demak. Akan tetapi saat ini jumlah vegetasi pohon mangrove mulai berkurang karena arus dan gelombang air laut yang kuat menyebabkan banyak pohon mangrove yang mati. Selain itu juga karena mulai berkurangnya perhatian masyarakat dalam upaya pemeliharaan mangrove. Seiring dengan berakhirnya proyek rehabilitasi mangrove oleh OISCA di Desa Bedono, semangat dan motivasi masyarakat mulai berkurang dalam memelihara mangrove. Banyak pohon mangrove yang roboh dan mati tidak lagi mendapatkan perhatian yang serius oleh masyarakat. Kesadaran masyarakat dalam menjaga keberlangsungan konservasi mangrove tidak merata. Jika kondisi tersebut dibiarkan, maka upaya konservasi mangrove tidak akan berkelanjutan dan kualitas lingkungan pesisir kembali menurun. Kelompok laki-laki masih lebih dominan dibandingkan dengan kelompok perempuan, baik secara personal maupun secara kelembagaan. Padahal kelompok perempuan merupakan kelompok masyarakat yang memiliki peran sentral dalam mereproduksi pengetahuan tentang mangrove (Laksono dkk., 2000). Begitupun dengan kelompok anak-anak, minimnya sosialisasi tentang konservasi mangrove dari keluarga berpotensi untuk menghambat upaya rehabilitasi mangrove. Hal ini berdasarkan hasil penelitian tahun 2020 sebelumnya, terkait literasi mangrove di lokasi yang sama. Para ibu-ibu mengaku tidak melakukan sosialisasi tentang konservasi mangrove dan bahwa sosialisasi tersebut seringkali dilakukan oleh mahasiswa dari perguruan tinggi atau lembaga-lembaga internasional. Padahal, agar konservasi lingkungan pantai melalui rehabilitasi mangrove di Bedono dapat berkelanjutan, maka diperlukan adanya kesadaran, partisipasi dan tanggung jawab penuh oleh berbagai lapisan masyarakat lokal (Luthfi, 2020). Dalam hal ini, literasi mangrove dan pengetahuan lokal masyarakat dapat dihimpun, direproduksi, dan diinternalisasikan kembali agar pemeliharaan mangrove menjadi bagian dari aktifitas sosial budaya mereka. Segmen masyarakat yang paling efektif dalam upaya literasi mangrove berbasis kearifan lokal ini adalah anak-anak sebagai generasi penerus di Desa Bedono. Melalui upaya internalisasi mangrove ke kelompok anak-anak, maka kesadaran konservasi mangrove dan kepedulian atas keselamatan lingkungan lingkungan dapat tertanam kepada masyarakat Bedono sejak dini. Praktik pendidikan konservasi kepada anak-anak sudah banyak dilakukan, namun dalam pelaksanaannya tidak sedikit berada di luar kesadaran. PENDAHULUAN Desa Bedono di Kecamatan Sayung Kabupaten Demak merupakan salah satu desa yang mengalami abrasi sangat parah dalam dua decade terakhir yang menenggelamkan sepertiga wilayah desa. Menurut data Kementerian Kelautan dan Perikanan tahun 2018, daerah pesisir di Kabupaten Demak merupakan daerah yang terkena abrasi cukup luas yaitu 798 hektar yang mengakibatkan banyak daerah hilang karena abrasi air laut. Daerah yang terkena abrasi di Demak meliputi 14 kecamatan, dimana Kecamatan Sayung terkena paling luas yaitu 250 hektar (Purbaya, 2017). Selain diakibatkan oleh arus dan gelombang air laut yang besar sebagai bentuk fenomena alam, degradasi wilayah pantai ini juga disebabkan oleh aktivitas manusia seperti petambakan, penambangan pasir, perluasan wilayah pemukiman, dan pembangunan infrastuktur di sekitar wilayah pantai (Damayanti, 2013; Tarigan, 2007; Raihansyah dkk., 2016; Suniada, 2015). Berbagai cara sudah ditempuh untuk meminimalisir abrasi pantai, seperti pembuatan sabuk pantai dan penanaman mangrove di pesisir pantai. Di Desa Bedono Kecamatan Sayung, Kabupaten Demak, upaya konservasi mangrove sudah lama diupayakan sejak wilayah pesisir ini dilanda abrasi pantai yang cukup besar yang mengakibatkan sejumlah pemukiman, tambak, dan areal pertanian 222 Abdimas Vol 26, No. 2 (2022): December 2022 METODE Pengabdian kepada masyarakat ini berlokasi di Desa Bedono, Kecamatan Sayung yang mana adalah daerah yang terimbas rob dan sebagian desa dalam posisi tenggelam. Berikut peta desa Bedono yang wilayah daratan sudah berkurang. Gambar 1. Peta satelit Desa Bedono, 2021. Gambar 1. Peta satelit Desa Bedono, 2021. Untuk merealisasikan tujuan pengabdian yaitu pelestarian lingkungan mangrove di masa depan, maka khalayak sasaran program pengabdian ini adalah masyarakat dan kelompok anak-anak di Desa Bedono, Kecamatan Sayung, Kabupaten Demak. Untuk merealisasikan tujuan pengabdian yaitu pelestarian lingkungan mangrove di masa depan, maka khalayak sasaran program pengabdian ini adalah masyarakat dan kelompok anak-anak di Desa Bedono, Kecamatan Sayung, Kabupaten Demak. Adapun metode yang digunakan adalah tiga tahap. Pertama, metode penggalian cerita rakyat. Metode ini mengawali keseluruhan kegiatan pengabdian dengan menggali cerota-cerita yang berkembang di masyarakat terkait dengan pemeliharaan mangrove dan menjaga lingkungan pantai. Tahap ini dilakukan dengan wawancara kepada beberapa narasumber dan sesepuh desa Bedono. Tahap ini dilakukan pada periode Agustus – September 2021 oleh tim mahasiswa. Kedua, penyusunan naskah komik. Tahap berikutnya adalah perumusan naskah komik dari data yang diperoleh pada tahap pertama tadi. Di sini dibuat storyline yang sesuai dengan karakter anak- anak. Tahap ini dilakukan oleh seluruh tim pengabdi dosen dan mahasiswa. Lalu setelah naskah selesai diserahkan kepada illustrator untuk dibuat cerita bergambar. Ketiga, sosialisasi dan story telling komik. Tahap setelah komik selesai dibuat adalah mensosialisasikannya kembali kepada masyarakat terutama kelompok anak-anak melalui story telling di sekolah-sekolah, taman bermain, taman alquran, dsb untuk mencapai tujuan internalisasi nilai konservasi mangrove. Abdimas Vol 26, No. 2 (2022): December 2022 diturunkan ke anak-anak dalam keluarga. Kearifan lokal merupakan modal sosial yang dimiliki masyarakat yang dapat menumbuhkan partisipasi masyarakat dalam merumuskan bersama bentuk pengelolaan sumber daya alam sekaligus mengelolanya Dengan secara sadar dan terstruktur praktik pengenalan pendidikan konservasi kepada anak-anak akan menjadi kunci dalam menjaga ekosistem kehidupan, termasuk di dalamnya adalah anak-anak manusia itu sendiri. Bukan hanya untuk hidup di masa ini, tetapi juga masa depan. Dengan demikian, diperlukan suatu kegiatan yang efektif bagi anak- anak untuk memahami nilai-nilai konservasi lingkungan dari kearifan lokal masyarakat mereka yang dituturkan melalui cerita rakyat. diturunkan ke anak-anak dalam keluarga. Kearifan lokal merupakan modal sosial yang dimiliki masyarakat yang dapat menumbuhkan partisipasi masyarakat dalam merumuskan bersama bentuk pengelolaan sumber daya alam sekaligus mengelolanya Dengan secara sadar dan terstruktur praktik pengenalan pendidikan konservasi kepada anak-anak akan menjadi kunci dalam menjaga ekosistem kehidupan, termasuk di dalamnya adalah anak-anak manusia itu sendiri. Bukan hanya untuk hidup di masa ini, tetapi juga masa depan. Dengan demikian, diperlukan suatu kegiatan yang efektif bagi anak- anak untuk memahami nilai-nilai konservasi lingkungan dari kearifan lokal masyarakat mereka yang dituturkan melalui cerita rakyat. Abdimas Vol 26, No. 2 (2022): December 2022 Artinya, praktik pendidikan tersebut merupakan bagian rutinitas tradisi dari proses pendidikan kepada anak-anak sebagai pewaris kebudayaan. Upaya konservasi budaya lewat sosialisasi kepada anak-anak menjadi peristiwa penting dalam pembelajaran anak (Rahmawati, 2012). Pun demikian dengan praktik pendidikan literasi lingkungan yang dilakukan kepada guru-guru dan kader posyandu dalam masyarakat Paledah (Pangandaran) yang dipandang mampu melahirkan manusia yang literat lingkungan. Hasil dari praktik pendidikan ini melahirkan para fasilitator pendidikan literasi lingkungan bagi anak-anak dan komunitas (Komariah dkk., 2017). Bagi masyarakat yang sudah sadar tentang hal itu sudah melakukan pendidikan konservasi secara terstruktur, seperti apa yang dilakukan oleh masyarakat Tengger. Masyarakat Tengger dengan kesadarannya melaksanakan pendidikan konservasi lewat lembaga formal dan kelompok masyarakat untuk meningkatkan keterampilan pengelolaan sumber daya yang tersedia (Sayektiningsih dkk., 2008). Hal serupa juga dilakukan oleh masyarakat Yogyakarta yang menerapkan strategi konservasi budaya dengan melibatkan semua elemen masyarakat (Soeroso dkk., 2008). Muara dari praktik pendidikan konservasi yang berbasis kearifan lokal dan kesesuaian konteks yang dilakukan oleh masyarakat adalah adanya perubahan pengetahuan, sikap, dan perilaku untuk pengelolaan sumber daya alam dan ekosistem yang ramah serta berkelanjutan (Fidela dkk., 2020). Kearifan lokal yang diwariskan secara turun temurun biasanya ditransmisikan melalui aktivitas domestik yang melibatkan perempuan. Sehingga internalisasi nilai-nilai kearifan lokal dapat 223 Abdimas Vol 26, No. 2 (2022): December 2022 Abdimas Vol 26, No. 2 (2022): December 2022 yang muncul di permukaan dengan bentuk yang tidak beraturan serta hanya bisa tumbuh pada air payau merupakan karakteristik utama pohon mangrove. Masyarakat di daerah pesisir sering menyebut mangrove dengan pohon bakau dengan beberapa nama lokal yang berbeda dari setiap wilayah. Masyarakat di pantai utara Jawa umumnya mengenal mangrove sebagai pohon yang gampang tumbuh, sebab buah yang jatuh dari pohon terkadang tumbuh menjadi tunas yang banyak. Hal ini membuat vegetasi mangrove alami banyak tidak dirawat secara khusus oleh masyarakat karena adanya anggapan di atas. Namun di sisi lain, pohon mangrove juga sangat rentan akibat terjangan ombak yang besar sehingga tidak sedikit tunas ataupun pohon mangrove yang sudah besar menjadi tumbang dan mati. g Di Desa Bedono, masyarakat pada awalnya memiliki asumsi seperti masyarakat pesisir pada umumnya yang menganggap mangrove sebagai pohon liar yang bisa tumbuh di sekitar rumah mereka tanpa harus ditanam dengan sengaja. Pohon mangrove yang tumbuh mereka beri nama yang berbeda sesuai dengan jenisnya, yakni Brayo, Bedada, dan Bakau. Ranting dari pohon mangrove yang sudah besar banyak dimanfaatkan oleh masyarakat sebagai kayu bakar, sedangkan getahnya digunakan sebagai obat luka. Tanaman Mangrove yang tumbuh di bibir pantai juga difungsikan oleh masyarakat sebagai tanaman yang bisa menahan gelombang air laut. Hanya saja, pada sekitar tahun 1980an, pohon mangrove mulai banyak ditebangi untuk perluasan areal pertambakan. Saat itu, nilai ekonomis dari udang windu dan hasil tambak lainnya sangat tinggi sebab banyaknya permintaan dari luar negeri. Ini membuat vegetasi mangrove di Bedono mulai berkurang. Kondisi ini diperparah dengan adanya terjangan ombak yang besar akibat pembangunan Pelabuhan Tanjung Mas di Kota Semarang. Pohon-pohon mangrove yang tumbuh di sekitar pantai tidak banyak yang bisa bertahan hidup akibat gelombang yang besar dan abrasi pantai yang terjadi terus menerus. Sejak akhir tahun 1999, vegetasi mangrove di Bedono semakin menyusut seiring dengan abrasi pantai yang bahkan membuat pemukiman warga menjadi tergenang. Bahkan beberapa Kepala Keluarga harus direlokasi karena rumahnya sudah tidak layak lagi dihuni sebab areal pemukiman mereka sudah menyatu dengan laut. Bencana abrasi ini membuat masyarakat mulai tersadar akan pentingnya mangrove untuk menjaga lingkungan mereka. Terlebih ketika makam dari seoarang ulama mulai tergenang dan hamper tenggelam. Hal ini membuat beberapa warga berinisiatif untuk menanam mangrove di sekitar makam tersebut dan tidak bersedia direlokasi agar dapat menjaga makam tersebut. Hanya saja, cara-cara penamaman dan pemeliharaan mangrove kurang mereka pahami sebab selama ini, mangrove dianggap pohon liar yang bisa tumbuh sendiri. Pelaksanaan Kegiatan Pengabdian g g Untuk membantu melaksanakan kegiatan Internalisasi Nilai-nilai Konservasi Lingkungan Melalui Penuturan Cerita Rakyat Bagi Anak-anak di Desa Bedono Kecamatan Sayung Kabupaten Demak, maka ada beberapa tahapan kegiatan. Abdimas Vol 26, No. 2 (2022): December 2022 Hingga pada tahun 2006, OISCA (The Organization for Industrial, Spiritual, dan Cultural Advancement-International), sebuah lembaga nirlaba dari Jepang, mengembangkan program rehabilitasi lingkungan pantai di Desa Bedono. Melalui program ini, masyarakat dikenalkan dan diajarkan cara membudidayakan pohon mangrove, mulai dari pembibitan, penanaman, perawatan, dan pemantauan. Saat ini, program rehabilitasi pantai dari OISCA telah membuat lingkungan pantai di Bedono hijau kembali dengan hutan mangrove yang cukup lebat.. HASIL DAN PEMBAHASAN Pengetahuan Lokal Masyarakat tentang Konservasi Mangrove Mangrove merupakan tanaman khas pesisir yang banyak dijumpai di sepanjang pantai. Akar Pengetahuan Lokal Masyarakat tentang Konservasi Mangrove Pengetahuan Lokal Masyarakat tentang Konservasi Mangrove Mangrove merupakan tanaman khas pesisir yang banyak dijumpai di sepanjang pantai. 224 Abdimas Vol 26, No. 2 (2022): December 2022 Tahap Perencanaan Pada tahap ini, tim pengabdi merencanakan konsep kegiatan yaitu menggali informasi dari perangkat desa dan sesepuh desa tentang cerita-cerita yang berkembang di masyarakat tentang nilai konservasi lingkungan, sejarah mangrove, dan manfaat mangrove bagi masyarakat. Melalui serangkaian wawancara dan focus group discussion ini, tim telah memperoleh gambaran tentang metode pelaksanaan kegiatan pengabdian. Dari cerita para tokoh masyarakat, diketahui bagaimana awal mula rob terjadi di Desa Bedono, datangnya berbagai lembaga yang membantu masyarakat dengan salah satunya menanam mangrove hingga manfaat mangrove bagi kehidupan masyarakat desa 225 Abdimas Vol 26, No. 2 (2022): December 2022 Gambar 2. Wawancara narasumber terkait penggalian cerita rakyat di Bedono saat ini. Gambar 2. Wawancara narasumber terkait penggalian cerita rakyat di Bedono Tahap Pelaksanan p Tahap ini adalah pembuatan komik berdasarkan cerita dari tokoh masyarakat dan sesepuh untuk dijadikan media komik untuk internalisasi nilai konservasi lingkungan untuk anak-anak. Komik ini bercerita tentang anak-anak Desa Bedono yang memperhatikan lingkungan rumahnya dan mencari tahu apa yang terjadi. Kemudian, keberadaan dan manfaat mangrove yang tumbuh di sekitar rumah mereka. Aspek terpenting adalah anak dapat belajar peka terhadap perubahan di lingkungan mereka, mencari jawaban apa yang terjadi, dan menumbuhkan kesadaran akan konservasi mangrove. Berikut adalah bagian dari komik yang dibuat. Gambar 3. Storyline komik Penyusunan storyline dilakukan oleh tim pengabdian dalam waktu satu bulan untuk memfinalisasi cerita yang akan dibangun dalam komik, termasuk pemilihan tokoh utama yaitu anak- anak agar mudah diterima oleh kelompok anak-anak yang menjadi target dalam pengabdian ini. Kemudian, cerita yang sudah disusun kemudian dilengkapi dengan ilustrasi gambar yang dikerjakan secara professional, hingga komik siap cetak. Tampilan halaman depan komik. Gambar 3. Storyline komik Gambar 3. Storyline komik Penyusunan storyline dilakukan oleh tim pengabdian dalam waktu satu bulan untuk memfinalisasi cerita yang akan dibangun dalam komik, termasuk pemilihan tokoh utama yaitu anak- anak agar mudah diterima oleh kelompok anak-anak yang menjadi target dalam pengabdian ini. Kemudian, cerita yang sudah disusun kemudian dilengkapi dengan ilustrasi gambar yang dikerjakan secara professional, hingga komik siap cetak. Tampilan halaman depan komik. 226 226 Abdimas Vol 26, No. 2 (2022): December 2022 Gambar 4. Halaman sampul komik yang sudah dicetak dan ber-ISBN. Gambar 4. Halaman sampul komik yang sudah dicetak dan ber-ISBN. SIMPULAN Kegiatan pengabdian yang dilakukan dosen UNNES ditujukan kepada masyarakat Desa Bedono khususnya kelompok anak-anak usia sekolah. Kerusakan lingkungan wilayah pesisir terjadi tidak hanya karena bencana alam saja, namun disebabkan juga oleh aktivitas manusia. Wilayah Desa Bedono, Kecamatan Sayung, Kabupaten Demak saat ini sudah kehilangan sepertiga wilayahnya karena abrasi laut. Penanaman mangrove sudah dilakukan, namun kesadaran masyarakat untuk memelihara dan melestarikan mangrove masih rendah. Dengan kegiatan ini diharapkan dapat menggugah kesadaran anak-anak untuk berperan dalam upaya rehabilitasi mangrove dan menjaga lingkungan. Bertambahnya ilmu pengetahuan bagi anak-anak secara khusus dan masyarakat Desa Bedono secara umum. Ilmu pengetahuan ini berasal dari kearifan lokal masyarakat lokal melalui penuturan pengalaman para sesepuh desa. Saran yang dapat disampaikan dari hasil pengabdian ini yaitu agar para orang tua di Desa Bedono melakukan internalisasi nilai konservasi kepada anak mereka sejak dini, sehingga dapat sadar dan melakukan Tindakan konservasi lingkungan terutama mangrove yang menjadi penyelamat wilayah Desa Bedono selama dua dasawarsa terakhir ini. UCAPAN TERIMA KASIH Tim pengabdi mengucapkan terima kasih kepada Universitas Negeri Semarang melalui Fakultas Ilmu Sosial atas dukungan pendanaan pengabdian kepada masyarakat tahun 2021 dengan nomor kontrak: 315.29.4/UN37/PPK.4.3/2021, tanggal 29 April 2021. Ucapan terima kasih juga disampaikan kepada masyarakat Desa Bedono terutama siswa SD Bedono 1 atas partisipasinya dalam kegiatan pengabdian ini. Tahap Evaluasi p Setelah pelaksanaan pengabdian terlaksana, maka tahap selanjutnya adalah tahap evaluasi. Tahap evaluasi dilakukan oleh tim pengabdi untuk mengetahui tingkat keberhasilan dari pengabdian yang sudah dilaksanakan. Evaluasi dilaksanakan dengan cara merefleksikan proses kegiatan pengabdian mulai perencanaan sampai pelaksanaan pengabdian. Selama proses evaluasi, tim pengabdi menilai bahwa pelaksanaan pengabdian berjalan dengan baik. Meskipun terdapat kendala utama dari kegiatan ini adalah masih merebaknya pandemic Covid-19 yang kemudian direspon oleh pemerintah pusat dan daerah dengan kebijakan PPKM. Kota Semarang dan Kabupaten Demak menjadi kota dengan level 4 atau tertinggi, sehingga pergerakan tim sangat terbatas. Selain itu, pengetahuan masyarakat yang terbatas terkait mangrove dan konservasi lingkungan sangat terbatas. Namun demikian, antusiasme siswa SD Bedono ketika sosialisasi dan pembacaan komik sangat tinggi sehingga ini menjadi indicator keberhasilan pengabdian di masyarakat juga. Bahkan pihak sekolah meminta agar berikutnya lebih terjadwal dengan jumlah siswa yang lebih banyak untuk mereka dapat mendengarkan cerita komik ini. Tim pengabdian juga menyerahkan 3 eksemplar komik untuk bahan pustaka di perpustakaan SD Bedono 1. Proses kegiatan ini dinilai baik karena dilaksanakan sesuai dengan rencana yang telah disusun Tim PPM dengan tahapan-tahapan yang dirumuskan. Evaluasi dilakukan bersama antara Tim PPM Unnes dengan semua pihak yang terkait, perangkat desa Bedono, Sayung, Demak. Hal ini bertujuan agar Tim PPM Unnes maupun peserta dan stake holder dapat mengetahui keefektivitasan kegiatan yang dilakukan demi tercapainya target pengabdian ini. Tahap Sosialisasi p Pada tahap ini komik yang sudah dicetak dan diperbanyak, kemudian siap dibagi-bagikan kepada pemerintah desa Bedono, sekolah dasar, sekolah keagamaan dan komunitas desa. Sosialisasi dilakukan dengan cara dating langsung ke kegiatan anak-anak yaitu di sekolah. Sosialisasi kepada anak-anak dengan cara story telling dan games yang dilakukan oleh tim mahasiswa. Pada tahap ini komik yang sudah dicetak dan diperbanyak, kemudian siap dibagi-bagikan kepada pemerintah desa Bedono, sekolah dasar, sekolah keagamaan dan komunitas desa. Sosialisasi dilakukan dengan cara dating langsung ke kegiatan anak-anak yaitu di sekolah. Sosialisasi kepada anak-anak dengan cara story telling dan games yang dilakukan oleh tim mahasiswa. Gambar 5. Sosialisasi komik di sekolah SD Bedono Gambar 5. Sosialisasi komik di sekolah SD Bedono Para siswa sangat antusias dengan kedatangan tim pengabdian yang membawa buku komik untuk mereka. Kemudian, tim pengabdian membacakan isi komik tersebut dan di akhir sesi pembacaan ada tanya jawab. Untuk menambah kemeriahan acara maka sesi tanya jawab para siswa diberikan kenang-kenangan berupa makanan kecil dan minuman. Dengan kegiatan menyenangkan ini, tim pengabdian juga menyerahkan beberapa eksemplar komik sebagai bahan pustaka di perpustakaan SD Bedono. Dengan demikian, siswa dapat sewaktu-waktu membacanya dan mendapatkan inspirasi tentang nilai konservasi, nilai persahabatan, dan motivasi untuk berprestasi sebagaimana pengalaman yang diceritakan oleh tokoh utama dalam komik tersebut. Gambar 5. Sosialisasi komik di sekolah SD Bedono Para siswa sangat antusias dengan kedatangan tim pengabdian yang membawa buku komik untuk mereka. Kemudian, tim pengabdian membacakan isi komik tersebut dan di akhir sesi pembacaan ada tanya jawab. Untuk menambah kemeriahan acara maka sesi tanya jawab para siswa diberikan kenang-kenangan berupa makanan kecil dan minuman. Dengan kegiatan menyenangkan ini, tim pengabdian juga menyerahkan beberapa eksemplar komik sebagai bahan pustaka di perpustakaan SD Bedono. Dengan demikian, siswa dapat sewaktu-waktu membacanya dan mendapatkan inspirasi tentang nilai konservasi, nilai persahabatan, dan motivasi untuk berprestasi sebagaimana pengalaman yang diceritakan oleh tokoh utama dalam komik tersebut. 227 227 Purbaya, A. A. (2017). 798 Hektar di Pesisir Demak Tenggelam AKibat Abrasi dan Rob. https://news.detik.com/berita-jawa-tengah/d-3481491/798--hektar-di-pesisir-demak- tenggelam akibat abrasi dan rob (Diakses tanggal 28 Februari 2020) gg gg Damayanti, K. (2013). Dampak Abrasi Pantai Terhadap Lingkungan Sosial (Studi Kasus di Desa Bedono, Sayung Demak), Pros. Semin. Nas. Pengelolaan Sumbar Daya Alam dan Lingkungan, Purbaya, A. A. (2017). 798 Hektar di Pesisir Demak Tenggelam AKibat Abrasi dan Rob. https://news.detik.com/berita-jawa-tengah/d-3481491/798--hektar-di-pesisir-demak- tenggelam-akibat-abrasi-dan-rob. (Diakses tanggal 28 Februari 2020). Damayanti, K. (2013). 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https://openalex.org/W2234703215	https://hal.science/hal-01341468/document	English	null	Characterization of two Austrian porcine reproductive and respiratory syndrome virus (PRRSV) field isolates reveals relationship to East Asian strains	Veterinary research	2,016	cc-by	23,011	© 2016 Sinn et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) causes major problems for the swine industry world‑ wide. Due to Austria’s central location in Europe, a large number of animals are transported through the coun‑ try. However, little is known about current PRRSV strains and epidemiology. We determined full-length genome sequences of two Austrian field isolates (AUT13-883 and AUT14-440) from recent PRRSV outbreaks and of a related German isolate (GER09-613). Phylogenetic analysis revealed that the strains belong to European genotype 1 subtype 1 and form a cluster together with a South Korean strain. Remarkably, AUT14-440 infected the simian cell line MARC- 145 without prior adaptation. In addition, this isolate showed exceptional deletions in nonstructural protein 2, in the overlapping region of glycoprotein 3 and 4 and in the 3′ untranslated region. Both Austrian isolates caused similar lung lesions but only pigs infected with AUT14-440 developed clear clinical signs of infection. Taken together, the genetic and biological characterization of two novel Austrian PRRSV field isolates revealed similarities to East Asian strains. This stresses the necessity for a more detailed analysis of current PRRSV strains in Europe beyond the determi‑ nation of short ORF5 and ORF7 sequences. type 1 (PRRSV-1), whereas VR2332 represents the North American PRRSV type 2 (PRRSV-2). The two genotypes share only about 60% identity at the nucleotide level [5]. Due to high mutation and recombination rates [6], vari- ability is also high within the genotypes, especially in type 1. Therefore, three subtypes have been proposed for PRRSV-1 based the size of the nucleocapsid protein (N): pan-European subtype 1 and Eastern European subtypes 2 and 3 [7]. Characterization of two Austrian porcine reproductive and respiratory syndrome virus (PRRSV) field isolates reveals relationship to East Asian strains Leonie J. Sinn1, Leonie Zieglowski1, Hanna Koinig2, Benjamin Lamp1, Bettina Jansko3, Georg Mößlacher3, Christiane Riedel1, Isabel Hennig‑Pauka2 and Till Rümenapf1 Correspondence: leonie.sinn@vetmeduni.ac.at; till.ruemenapf@vetmeduni.ac.at 1 Institute of Virology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria Full list of author information is available at the end of the article Veterinary Research Veterinary Research Veterinary Research Sinn et al. Vet Res (2016) 47:17 DOI 10.1186/s13567-015-0293-x Open Access © 2016 Sinn et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium 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. Virus isolation and titration For virus isolation, 50 µL serum of diseased piglets from farms 440 and 883 were used to inoculate 5 × 106 PAM on a six-well format. After 2 days, the cell culture super- natant was clarified by centrifugation (5 min, 3000 g) and passaged on PAM and MARC-145 cells. Virus infection was generally analysed 2 days post-infection by immuno- fluorescence staining. Virus titres of supernatants were confirmed on PAM from a single animal to ensure com- parability and expressed as tissue culture infectious dose of 50% (TCID50). In the animal trial a viral stock from the 6th to 8th passage was used for inoculation. f The aim of this study was to determine the etiological agents of two PRRSV-1 outbreaks in Austria and to char- acterize them genetically and biologically. The results reveal that the isolated PRRSV-1 subtype 1 field strains (AUT13-883 and AUT14-440) have genetic and phyloge- netic similarities to East Asian strains. Introduction This gap in knowledge complicates the identification of the source of outbreaks, which is crucial as the most important PRRSV problems in Austria occur as a con- sequence of (re-) introduction into formerly free herds. An awareness of recent isolates and genomic changes that possibly lead to lower protection is also important in terms of assessing the effectiveness of vaccines. streptomycin). Two additional antibiotics (enrofloxacin and kanamycin) were added for the culture of PAM. All cells were maintained at 37 °C and 5% CO2 and observed daily for cytopathic effects. Materials and methods Viruses and cells Wild-type PRRSV strains AUT13-883 and AUT14-440 were isolated from sera of two farms in Upper Austria in 2013 and 2014. A cell culture adapted PRRSV-1 sub- type 1 strain (GER09-613) that was isolated from the field in Germany in 2009 was also included in the analysis (kindly provided by L. Haas, TiHo Hannover). MARC- 145 cells (CCLV RIE 277 [18]) were obtained from the Friedrich-Loeffler-Institute in Germany. Preparation of porcine alveolar macrophages (PAM) was essentially per- formed as described previously [19]. Briefly, lungs of 6- to 12 week-old PRRSV-free, healthy pigs were washed 2–3 times with PBS (pH 7.2). Aliquots of lavage fluid were pooled and centrifuged for 5 min at 1000 g. The cells were washed in PBS and repelleted twice. For storage, PAM were resuspended in FCS supplemented with 10% dime- thyl sulfoxide and frozen at −150 °C. MARC-145 cells were cultured in Dulbeccos’s modified Eagle’s medium (Life Technologies, Waltham, USA) supplemented with 10% fetal calf serum (Bio&Sell, Nürnberg, Germany) and antibiotics (100 U/mL penicillin and 100 µg/mL Sample collection Serum samples were collected from two medium-sized piglet producers in Upper Austria in November 2013 (farm 883) and March 2014 (farm 440). Both farms were experiencing PRRSV-related problems such as pneumonia, wasting, variance in growth, conjunctivitis and coughing in rearing piglets, as well as reproduc- tive disorders such as mummification and abortions in sows. Whereas one farm (883) had experienced minor problems related to PRRSV over a period of 2 years, the other (440) was formerly free of PRRSV and had losses of up to 50% in one farrowing badge. Serum samples from both farms had been tested positive for PRRSV by qRT-PCR by the Animal Health Service Upper Austria. Neither of the farms had used vaccines against PRRSV. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens in the swine industry worldwide [1, 2]. It is the etiological agent of porcine reproductive and respiratory syndrome (PRRS), which is characterized by respiratory disorders as well as by growth retardation in growing pigs and reproductive failure in late gestation sows [3, 4]. The disease emerged in the late eighties in North America, with the first outbreaks in Europe recorded in 1990 [3]. PRRSV is a small, enveloped virus with a single- stranded positive-sense RNA genome that belongs to the family Arteriviridae, order Nidovirales [8]. The 5′-capped and 3′-polyadenylated genome of PRRSV is about 15 kb in length and contains ten open reading frames (ORF) [9, 10]. ORF1a and 1b constitute over 75% of the viral genome and encode two polyproteins, which are cleaved into at least 14 nonstructural proteins (nsp) that are responsible for genome replication and transcription i The causative agent, PRRSV, was first isolated in 1991 in the Netherlands [4]. This strain, Lelystad virus (LV), is regarded as the prototype strain of European PRRSV Correspondence: leonie.sinn@vetmeduni.ac.at; till.ruemenapf@vetmeduni.ac.at 1 Institute of Virology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria Full list of author information is available at the end of the article Page 2 of 13 Sinn et al. Vet Res (2016) 47:17 [11]. ORF2–4 encode the minor structural proteins, including glycoprotein (GP) 2, 3 and 4, which form a hetero-trimer that is believed to be involved in virus entry [12]. The major structural proteins GP5, membrane protein (M) and N protein are encoded by ORF5–7. Nucleotide sequences of ORF5 (603–606 bp) and ORF7 (372–387 bp) are widely used for phylogenetic studies. However, the analysis is limited as these short genomic sequences might be subject to recombination and immu- nological selection pressure [6]. Phylogenetic analysis based on ORF5 and ORF7 may also lead to different sub- typing of PRRSV-1 strains [13]. For these reasons, the use of further methodologies for genetic subtyping has been suggested, such as whole genome sequencing [6, 14]. The lack of published full-length sequences is exemplified by the situation in Austria, where the limited data on current strains consists solely of ORF5 or ORF7 sequences [15– 17]. Detection of PRRSV‑positive cellsi Cells were fixed with 1:2 methanol-acetone for 2 min at room temperature and air-dried. Anti-PRRSV-N-protein monoclonal antibodies (clone P10/b1) [20] (kindly pro- vided by A. Saalmüller, Vienna) were employed for the detection of infected cells. Goat anti-mouse conjugated with Cy3 (Dianova/Jackson, Hamburg, Germany) was used as a secondary antibody. Quantitative reverse transcription‑PCR (qRT‑PCR) Quantitative reverse transcription PCR (qRT PCR) To detect viremia, viral RNA was extracted from 140 µL serum with QIAamp Viral RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted in 60 µL distilled water and 1 µL was directly used for amplification with the KAPA™ SYBR® FAST One-Step qRT-PCR Kit (Peqlab, Erlangen, Ger- many) on an ABI 7500 cycler (Applied Biosystems, Foster City, USA). Published primers Pesch PLS (5′-ATGGCC AGCCAGTCAATC-3′) and Pesch PLR (5′-TCGCCCT AATTGAATAGGTG-3′) were used for amplifica- tion [22]. A recombinant cDNA clone of a PRRSV-1 strain was purified using the QIAGEN Plasmid Midi Kit (Qiagen, Hilden, Germany) and spectrophotometri- cally quantified. The copy number of recombinant plas- mids was calculated with the formula: N (molecules per µL) = [C (DNA concentration in µg/µL)/K (fragment size in bp)] × 185.5 × 1013 (factors derived from DNA weight, volume and the Avogadro constant). To obtain a standard curve, a tenfold dilution series of cDNA of the PRRSV-1 clone was included in the qRT-PCR setup. Cycling condi- tions were 42 °C 5:00, 95 °C 5:00 and 42 cycles of 95 °C 0:03, 60 °C 1:00 and 80 °C 0:33 (fluorescence detection step) followed by a dissociation step (95 °C 0:15, 60 °C 1:00, 95 °C 0:15). The numbers of genome copies were calculated with the 7500 System SDS Software (Applied Biosystems, Foster City, USA) based on the standard curve and projected to 1 mL serum by multiplication by 428.4. The DNA fragments were subjected to gel electro- phoresis, purified by the peqGOLD Gel Extraction Kit (Peqlab, Erlangen, Germany) and sequenced by com- mercial laboratories (Microsynth Austria, Vienna, Aus- tria and Eurofins Genomics, Ebersberg, Germany). Primer selection for sequencing was based on available PRRSV-1 sequences. The determined full-length genome sequences of GER09-613, AUT14-440 and AUT13-883 were submitted to GenBank (KT344816, KT334375 and KT326148). Initial phylogenetic analysis was carried out with NCBI’s Basic Local Alignment Search Tool for nucle- otides (BLASTn). The two closest neighbours of the isolates presented in this study were determined for ORF5 and ORF7. Pairwise comparison and identity calculations were carried out with CLC Main Work- bench 7.6 (CLCBIO, Aarhus, Denmark). Alignments and phylogenetic trees were generated with the soft- ware CLC Sequence Viewer 7.6 (CLCBIO, Aarhus, Denmark) with bootstrap values based on 1000 repli- cates. All PRRSV-1 strains with full genome sequences deposited in GenBank were used to construct the phylogenetic trees. Determination and analysis of full‑length genome sequences Total RNA was extracted from PAM 5 days after inocula- tion and from serum samples using the RNeasy Mini Kit and the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted in 30 µL distilled water and either used immediately for RT-PCR or stored at −80 °C for subse- quent analysis. Page 3 of 13 Sinn et al. Vet Res (2016) 47:17 RT-PCR was carried out using the LongAmpKit (NEB, Ipswich, USA) or the One Step RT-PCR Kit (Qia- gen, Hilden, Germany) according to the manufacturer’s instructions. Seven primer pairs that were highly con- served in PRRSV-1 strains were designed (available upon request) based on published sequences available in NCBI GenBank. The resulting PCR amplicons were overlapping and covered the whole genome. Serum titration Tenfold dilution series of sera were prepared and trans- ferred to naïve PAM. For each time point and each ani- mal, two replicates were performed. Three days later cells were fixed and stained and the TCID50 was calculated. Quantitative reverse transcription‑PCR (qRT‑PCR) For the trees based on ORF5 and ORF7, the two closest neighbours—as determined by NCBI BLASTn—were added. The PRRSV-2 prototype VR-2332 was used as an out-group. Recombination analysis was performed with the recombination analy- sis tool (RAT) [21] using the full genome alignment of PRRSV-1 strains. To detect viral shedding, nasal swabs were submerged in 2 mL of sterile saline (0.7% NaCl) and thoroughly shaken for 30 s. 200 µL of supernatant were used for viral RNA extraction with the High Pure Viral RNA Kit (Roche Diagnostics, Vienna, Austria) according to the manufac- turer’s instructions. RNA was eluted in 50 µL of the kit’s elution buffer and 13.6 µL were directly used for ampli- fication with the LightCycler® RNA Amplification Kit SYBR Green I with a LightCycler® 480-II System (Roche Diagnostics, Vienna, Austria). The nested RT-PCR from Pesch [21] was adapted for detection with SYBR Green I®. Reverse transcription and activation was performed according to the manufacturer’s instructions, followed by 32 cycles of 60 °C 0:10, 72 °C 0:22 and 95 °C 0:10. The nested amplification step was carried out with LightCy- cler® FastStart DNA Master(plus) SYBR Green I (Roche Diagnostics, Vienna, Austria) using 2 µL of the first PCR reaction. Cycling conditions were 25 cycles of 60 °C 0:10, Animal trial Animals Twenty-seven-week-old conventional pigs (#1–20) were obtained from a PRRSV-negative herd and housed in a biosafety level 2 facility. They had been vaccinated against PCV-2 and M. hyopneumoniae in their third week of life. To confirm their PRRSV status nasal swabs taken at the time of arrival were analysed by qRT-PCR and serum samples were tested for antibodies against PRRSV with the commercial IDEXX X3 ELISA (IDEXX laboratories, Westbrook, USA). The animal experi- ments were approved by the ethics committee of the University of Veterinary Medicine, Vienna and the Federal Ministry of Science, Research and Economy (BMWF-68.205/0196-WF/V/3b/2014). Page 4 of 13 Sinn et al. Vet Res (2016) 47:17 Korean strain KNU-07 [24], a Croatian strain [25] and German strains [26] (Additional file 2). Korean strain KNU-07 [24], a Croatian strain [25] and German strains [26] (Additional file 2). 72 °C 0:20 and 95 °C 0:20. The fluorescence detection step was carried out at 84 °C. The PCR was followed by a dis- sociation step. Results were considered positive when a clear amplification curve was observed and the melting temperature of the amplicons (calculated by LightCycler® 480 System Software) was within the expected range. i Since the results of the phylogenetic trees based on ORF5 and ORF7 were inconclusive, complete genome sequences for AUT13-883 and AUT14-440 were deter- mined (GenBank: KT326148 and KT334375). A phylo- genetic tree based on the full genome sequences of the isolates presented in this study, 38 published PRRSV-1 strains and PRRSV-2 prototype VR-2332 (Figure 1) confirmed the relationship between the two Austrian isolates, GER09-613 and KNU-07. All four strains clus- ter together, with no other strain in the same branch. AUT13-883 and GER09-613 are the most closely related strains. Serology Serum samples were tested for antibodies against PRRSV with the commercial IDEXX X3 ELISA (IDEXX labora- tories, Westbrook, USA) according to the manufacturer’s instructions. Ratios of optical densities of sample and positive control (P/PK ratios) were calculated and con- sidered positive when >0.4. Statistical analysis Assembly of the overlapping sequences resulted in com- plete genomes consisting of 15022 nucleotides (nt) for AUT14-440 and 15095 nt for GER09-613 and AUT13- 883, excluding the 3′ poly(A) tails. Detailed comparison to the European prototype LV revealed marked differ- ences in the region of nsp2. Both Austrian virus isolates and the German strain GER09-613 possess a shorter ORF1a due to deletions in nsp2 (Figure 2). The differ- ence of three nucleotides in ORF1a between AUT13-883 (7188 nt) and LV (7191 nt) results in the deletion of pro- line 182 in nsp2 (Figure 3A, dotted line box). The deletion is also present in the South Korean strain KNU-07 and has been described for strains from Hong Kong. GER09- 613 and AUT14-440 (ORF1a lengths of 7188 and 7152 nt) share a deletion of three nt, resulting in the loss of glutamine 183 in nsp2, one amino acid (aa) downstream of proline 182 (Figure 3A, dotted line box). In compari- son to LV, AUT14-440 has further deletions in nsp2 at aa positions 320–323, aa 359–364 and aa 699–700, all in hypervariable regions of nsp2 where deletions are frequently found (Figure 3A, solid line boxes). ORF3 and ORF4 of AUT14-440 were also truncated by 36 nt when compared to LV, resulting in a deletion of 12 aa in the overlapping region of GP3 and GP4 (Figures 3B and C solid line boxes). Shorter deletions (up to 8 aa) in this area have been described for several strains, including HK5 and the Chinese isolate BJEU06-1. AUT14-440 also shows a single nucleotide deletion at position 87 in the 3′ untranslated region (UTR) (Figure 3D, solid line box). All deletions in virus isolate AUT14-440 were also found when RNA from the original serum sample was used for RT-PCR and PCR products were directly sequenced. Data were analysed with the SPSS statistics software (SPSS Ltd, Quarry Bay, Hong Kong). Oneway ANOVA with Posthoc LSD test was applied for the analysis of all results except for those from the ELISA, which were ana- lysed with the t test with the test value 0. A p value < 0.05 was considered statistically significant. Virus isolation Two PRRSV-1 outbreaks in 2013 (farm 883) and 2014 (farm 440) were brought to our attention by the responsi- ble veterinarians. Case 883 was characterized by mild res- piratory disorders and a low virus load that persisted over 2 years. In case 440, suckling piglet mortality reached 50%, which is severe for PRRSV outbreaks in Austria, and abortions were observed. Both farms had been found to be PRRSV-1 positive by qRT-PCR before we obtained any samples. To investigate the PRRSV strains responsible for the outbreaks, we isolated two viruses from the serum of diseased piglets after serial inoculation of PAM (termed AUT13-883 and AUT14-440). MARC-145 cells were also susceptible for AUT14-440 without adaption but were not infected by AUT13-883. Phylogenetic analysish The sequences of the ORF5 and 7 genes revealed that both isolates belong to PRRSV-1 subtype 1 [7] but the results for ORF5 and ORF7 were not consistent. In the phylogenetic tree based on ORF5, AUT14-440 clustered together with German and South Korean PRRSV-1 iso- lates (among them KNU-07) (Additional file 1). AUT13- 883 represented a different branch of the tree and was most closely related to German strain EU-2a from 1992 [23] and to Austrian strain 2888 [15]. In contrast, when ORF7 sequences were used for phylogenetic analysis, the two Austrian isolates formed a group together with Pairwise nucleotide comparison of the complete genome resulted in identities of 87.7, 87.9 and 88.6% with LV for AUT14-440, AUT13-883 and GER09-613. Identities between the isolates were 86.4% (GER09-613 Page 5 of 13 Sinn et al. Vet Res (2016) 47:17 Figure 1 Phylogenetic analysis based on nucleotide sequences of full-length genomes of 41 PRRSV-1 strains and PRRSV-2 prototype VR2332 as an out-group. The PRRSV strains presented in this study are marked in red and the associated sub-tree is highlighted with a box. The tree was constructed using the neighbour joining method with the numbers at the nodes representing bootstrap values in % of 1000 replicates. Scale bar: number of substitutions per site. Figure 1 Phylogenetic analysis based on nucleotide sequences of full-length genomes of 41 PRRSV-1 strains and PRRSV-2 prototype VR2332 as an out-group. The PRRSV strains presented in this study are marked in red and the associated sub-tree is highlighted with a box. The tree was constructed using the neighbour joining method with the numbers at the nodes representing bootstrap values in % of 1000 replicates. Scale bar: number of substitutions per site. to AUT14-440), 85.4% (AUT14-440 to AUT13-883) and 89.9% (AUT13-883 to GER09-613) (Figure 2). been described. However, the particular clinical signs in an affected herd provide no more than an indication of the pathogenicity of a PRRSV isolate, as other patho- gens may be involved. To assess the virulence and to test Koch’s postulates, an animal trial was performed with the Austrian isolates AUT13-883 and AUT14-440 and the German isolate GER09-613, which clusters in the same branch of the phylogenetic tree. Determination of virulence Genetic and phylogenetic analysis showed a striking relationship between the Austrian isolates, the German strain GER09-613 and the South Korean strain KNU-07. The reported severity of the clinical symptoms associ- ated with AUT13-883 and AUT14-440 differed markedly, while the clinical symptoms of GER09-613 have not PRRSV-seronegative pigs were distributed equally into four groups according to their weight and sex. Each Page 6 of 13 Sinn et al. Vet Res (2016) 47:17 Figure 2 Detailed comparison of complete genomes of GER09-613, AUT14-440, AUT13-883 and Lelystad virus (LV) (table) and schematic overview of nucleotide differences between LV and the isolates presented in this study. Black bars represent areas with a low degree of similarity to LV. Open reading frames (ORF) of PRRSV that encode non-structural proteins (blue) and structural proteins (orange) are shown. group of five animals was housed in a separate com- partment to avoid cross-contamination. After 1 week of adaptation (day 7 to day 0), pigs from groups GER09- 613, AUT13-883 and AUT14-440 were inoculated intranasally with 3 mL medium (1.5 mL in each nos- tril) containing 1 × 105 TCID50 of the respective strain. Pigs from the control group received the same amount of medium without virus. Clinical signs and rectal tem- perature were monitored daily. A scoring system was applied that rates liveliness, dyspnoea, coughing, nasal and ocular discharge, conjunctivitis and cyanosis with scores from 0 (physiological) to 3 (severe clinical signs). Daily scores for each animal were added to obtain a value for the overall health status and the mean clinical scores for the four groups were calculated (Figure 4A). All pigs infected with AUT14-440 showed mild to mod- erate dyspnoea on three or more study days. Other clinical signs that occurred frequently in all animals in this group were apathy and conjunctivitis. Two waves of clinical signs could be distinguished: the first wave occurred around day 4 post infection (pi) and the sec- ond, with more prominent signs of illness, took place around day 12 pi. In the other infection groups, only one (AUT13-883) or two (GER09-613) animals showed mild dyspnoea for no more than 2 days in a row. Pigs in the control group showed no relevant clinical signs in the trial, except for animal #4 that had to be euthanized on study day 10 due to a severe lameness in the hind legs. Determination of virulence There was no significant difference in rectal temperature between the four groups. The pigs were weighed at −7, 0, 3, 7, 10 and 13 days post infection (dpi). For each week of the trial (study days −7 to 0, 0 to 7, 7 to 13) daily weight gain was cal- culated and is shown for each group as a box plot with maximum and minimum values represented as whiskers (Figure 4B). From the first week to the second week of the trial, all groups showed an average increase of daily weight gain between 0.08 and 0.16 kg. From the second to the third week, the control group and group GER09-613 showed a mean increase of daily weight gain of 0.05 and 0.11 kg, whereas in the groups infected with the Austrian field isolates daily weight gain did not increase (mean values of −0.07 kg for AUT14-440 and −0.02 kg for AUT13-883). Despite the apparent differences between the groups, the figures are not statistically significant due to the small group size and the high variation within the Page 7 of 13 Page 7 of 13 Sinn et al. Vet Res (2016) 47:17 SG I AD F L AN P P PQE FWT L D KM L T SP SP ER SG F S S L YK L L L EV V PQK CGA T EGA F I YA V ERM L KD C P S SKQAMA L L A K I K V P S SK A P SV S L D EC F PT D V L AD F E PA SQE . . . . . . . . D . . . - . . . . . . . . . . . . . . . Q . . . . . . . . . . . . A . . . . . . . . K . . . T . . . . . . . . . . . . . E . . . . . . . . . . . . . . . . A . . . . . . . . . . . A P . N . . L . P . D A . . . . . . . . . . . - . R . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . . . V . . . . . . . . . . . . . D . . . . . . P . . L . . . P . . . . . S . . . . . . . - . . . . . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . R . . . . . . S . . . . . . . . . A . I S . . L . L . . . . . . . . . . . . D . . - . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . P . . L . L . P . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . P . . . . . . Determination of virulence F P . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N S . . . . . . A . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . S . . . . . T FH . . . . V . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . S . . . . . . . . . . . . P . N S . . . . K K . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . E . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . S . . . G . . . . D . . . . . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . P E . . . . . . . . . . . . . . . . . . . . . . . . . . AG . P . . . . . . F . . R PQ S SGA A V V L C SPD A K E F E EA A P E EVQE SGH K A VH SA L L A EGPNN EQVQV V AGEQ L K L GGCG L A VGN AH EGA L V SAG L I N L VGGN L SP SD PMK ENM L N SR ED E P L D L K . . . . . . . . . . . . . G . . . . . . T T L . . A . . . . R . V . Q . . . P . . . . . . . . ERT . . . . . . G . . . . . . V . . . . . . . T . . P . S . . D . G . R . . L F . N S . . . . V . . GWG . . . . . . . . . D P SV . A . P . P S . . . . . K . V . . . . . . D KD R . V . C PV P . S- - - - . K . . . M . T D . . . . . D SH . . L . E ST RK . . SA P . . . V - - - - - - V . S L . S . E . . T . . GW. Determination of virulence . . . . . . K . . . . D T . . A . . . . V V . . . . . . T L . . G . GGD Y . T . Y . . P . SD . . T . D . . . . A V . . . P . . D S . S SV A . . V . . . V . . PV . . . D SA SE S S L L L . S . . . . . F . . . GG . . . . . K . . . . . T V . . . R . . G . . . . . . . . L . . . . . . . . . T . . . . PH T K . . . K . . . . . . V . . . . . . . . . D . V . . . . . . D V . . . . . P . D . A S - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . N . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V P . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . T R . . . . . . . . . . . . G . . . . . . . . . . . . . L . . . . A . . . . . A . . . . . S . . . . . . . . . G . V . . . . SP . Determination of virulence . . . . E . . P . . . S . . G . . P . GW. . . . . . . . S . G . . T . G . S . . . . . . R . . K . T . . . . . . D . . E . . R . T P FT DD . - - - - - - - - - - - - - - - - - - - - - - - - - - - - T . A . . . ST . P . D . . . . S . . SA E . ST . . . . . . . . . . . N L . R . . . . . . S . . . . . . G SG . S . L . . I R . . D L . . . . . . . . . . D . . . . . . RA . . . . K P E . S . . N S . . . V . . . . V . A . V . . - - - - . . S . . . . G S . R . . I P . . WR . . . . . . GT . N . . . . . A . Y . . N V . . . . . . . . R . T . . GN . . . D . . . . P . R . . D EK . . . . . . . . . . E P S . . . S . . . . V . K . V . A . S . P . . . I S . D . P SPG F . . . D T . . . . . . . . . . . . . . . P . . . . A . . . . . . . G . . GT . S . . A . . . . . . . . . A V P . . . . . . . . . . . . . . . . . . E . . . . . . Determination of virulence . I . S . Q - - - - - - - - - - - - - - - - - . S . . SK R . . . H . . . . . . . . . . SQPA PA ST T T L V R EQT PDN PG SD AGA L PV T V R E FV PT GP I L CH V EH CGT E SGD S S SP L D L SD AQT L DQP L N L S L A AWPV RA T A SD PGWVHGRR E PV FV K PRN A F SDGD . . . . . T A . . . . . . . R . . . . . S F . . . . F . . . . . Q L A S . R SM . R . . . . . . A . . . . . . . . . . . . . . . A . . . . . D . . . . . . . . A . . . . . . . . I . . . . . . I . . . S . . V L . . . . . R . . ST A I A . A . K . . . . N . . . P EV I . S L A . . . . . . SA V . V . G . . G . . . M . R S . C . . . . . . . N T . . P . E . . . . . . . . . . . K T . . . . . . . . . . . . . . . . . . . . S . L . . . . . . S . S . A V . . P . E . H . . E . . . R . . . . F S . . T . R . . S . . FM . R . . . . . . . . . . . GN . . . . . . N . . A S . . . . . . . . T . . . . K . . . . . . . Determination of virulence . . . . . . . . . . . . . . D . . . . . . . R . . . . A . . . P EG . H . . E . . . F . . . . . L . . T . K . . . A . LM . H . . . . . . . . . . . G . . . . . . . . . . I PG . . . D . . . . . . . . K S . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . L T A . . . . . . . . . . . . . . . G . . . . . . . I . . . . . . R . . PR . . . . . . . . . S . . . . . . . . . . . . . P . . . . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . . . . G . . . . . . . . . . . V V . . G . . . . . . . CD L . F . . . . P F . . . . K . . . I . ST FR . . . . . . . . . DN I . . . . T . P . . Determination of virulence . A . . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GT L . . . . . . . T . V A . MA C . K . . I . Y . L . . . T P S- . . . AQ . . A . A S LM . R . . . . . . . . . . . . . . . . . . . N . . I . . . . . . . . . . T . . . K . . . . . . . . . . . . . . . . . . . . . S . L . . . . . . S . . . A . V . . . . G L . . . Y . . . N V . . P . . . - - - - A . . . L R FH . . . . . . . . P . . . . L . . . . . V . . A . N . . . D . . . T D . . . K T . . . . . . . . Y . . . . . . . . . R . D . . . . . . . H . . . . A . . . . . G . . . . . . . . . . . S . . . I A . . G . . . . . . . . R . . . . . . . . . . . . . . . . . . . F . . . . . . . . D . . . . . . . . K . . . . . . . . . R . . C . . . . L . . . K . . . . . . Determination of virulence SA L Q FGE L SE S S SV I E FD RT KD A PV VD A PVD L T T SN EA L SV VD P F E FA E L K R PR F SAQA L I D RGGP L AD VH A K I KN RV Y EQC L QA C E PG SRA T PA T R EWL D KMWD RVD . V . . . . . . F . . . . . V . . . . M . . . . . . . T . . . . . . . . K T P . A A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K K . . . . . . . . . . . . F . . . . F . . . . . . V . . . . A . N V . . . . . . . . . . . . . . T . . A . . . . . . . . P . . . . . . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . K R . . . . . . . . . . . V . . . . . T . . . . . . . . S . . A . . T . A A . I . I . . . . . S . . . F . I . . L . P . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . K K . . . . . . . . . . . V . . . . G . . . Determination of virulence . . . . . . . . . . . . T L . . . . . I . . . . . . . . . . M . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q . . . R . . . . . . . . . . . . . . . K K . . . R . . A . . . . . . . . . . . . . . . P . . . . . . . . . . . G . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . G I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . A . . . V . . . . L R E I . A . . . . . . . . P . . . . . . AD . . . R S . . F . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . Determination of virulence . . . . . . . . . K A . . . . . . . . . . . V . . L . . . . . . . . . V . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . A . . . . K . S . PR . . . . . . . . . . . . . . . . . E . . . . . . S . . . . . . . L . . . . . . . . . . . . K . . . . R . . . . . . . . . . . . . . . . . . . . . . . . Q . . . T L . A . . . . . . . . . . . . . . A . . . S . . V . . R . . . H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence MK TWRCT SQ FQAGR I L A S L K F L PDM I QD T P P PV PRKN RA SDN AG L KQ L V AQWD RK L SV T P P PK PVGPV L DQ I V P P PT D I QQED V T P SDGP PH A PD F P SRV ST GG SWKG . . . . . . . . . . . . . Q . . . . . N . . . . . . R . . . . . . . . . . . VN . SV . . . . . . . . . . . . . . . . . . Q . L . E . . . . . T I . . . . . A . . G . AN . P . R . . . . . . S . G . . G . SRG . . . . . . . H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . V S . S . . . . MM . . . . . . . E . L . Q . L . R . . P . . T G . S . V EV R . . . I P . T N E . . . V . . C - - . . G . . RN . R . . . . . C . . . . . . . . . . . . . . S . . . . . . R . . . . . . . . . . . . . . SV . . . . . . . . . . G . . . . . . . K . L . K . . . . . T I S S . . SA . . G . . . . P . E . . RV . . L SG . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . H . R . S . . . . R . . . . . . . . . . . . . L QR . A E . . P . . . A . S . MGV . . DG . . . . S . . . Q . . . . . G . . D . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . L . . . . . A . . . T . . . . . . . . . . . . . . . . R . . . V . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . T . G S . . . . . . . . . . . . . . . A . SQK . . . E . MRN . A T . . . . . . . . . E . . . P . . S . . . . . . . . . . . M SWN . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . D . . R . . . R . . . . . . A A S S . . . A . . . . G . T . . . . AN T . . . . . I . . . . . . . . . V . S . . . . M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . N . . . . . . . . . . . . . . . . S . . . Q . . . . . . . A . T . L . . MGT . . . . . . . . . . . . Q . . . L F . . . . . S . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . S . . . . . . . . R . . K . . . . . . . . . SA . L . . . . T . . . . . . . . . . . A . . . . . L S . . S . . S . . . . . SW. . . . MA A A T L F F L AGAQH I MV SEA FA CK PC F ST H L SD I ET N T T A A AG FMV L QD I N C FR PHGV SA AQEK I S FGK S SQCR EA VGT PQY I T I T AN V T D E SY L YN AD L LM L SA C L F YA . . T . I . . L . V . . . C L . . . . . . . Determination of virulence . . . . . . . . . . . . K . . . . . . . . . . . . . K . E . L Q . R . D . T T P SGV . YA . Q . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . L L . V . . . . L . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . K . E . L Q . - - - - - - - - - - - - K V R . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . L . V . . . R L V . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . K . E . SQ . . KN . ST SHGA PQT . R . . . . Q . I . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . I . . L . V . . . R L . . . . . . . . . . . . . . . . . . . K . . . . P . . N . . . . . K . E . L Q . . RG . T T . Q SV P YK . Q . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . L . . . . . Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . E . P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . I . . L . . . . . Y L . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . L . . . . . . T . . . T . . . . . P . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . LMV . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . G . . N . D . L . H . R . T . . . GR . YT RGP . . . . . V . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . L . V . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . L . . . . . T . AQ S L R . . - - - - - - - - R . P P . . Determination of virulence . . . . . I . . . . . . M . . . . . . . . . . . . . . . . . . . . . . . . . V . . I . . L . V S . . Y L V . . . . . . . . . . . . . . . . N . K . . . . . . . . . L . . . . . T . AQ S . V A . - - - - - - - - S . P . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A F . L . V . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . N . . . S . . . EA . . T . GQV P SR . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T GHN I SA L YA A Y YHHQ I DGGNWFH L EWL R P L F S SWL V L N I SWF L RR SPV SPV SRR I YQ I L R PT R PR L PV SWS FRT S I V SD L T G SQQRK RK F P SE SR PN V V K P SV L P ST SR . N Y . . . . S . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . V . . . . . . . . A . . A . . . . . . . . . . . . . . . . . . . . . K K L N . FN . A . T . Determination of virulence . . R . A . . MRN SR . . A . L . A . . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . L . A . . A . . . . . . . . . . . . . . . . . . . . . . K L N A . N P - - - - - - - - - - - - R YGH S . . . . . . . . . . L Q VN . . . . . S . . . . . . . . . . . . . . . . . . . . . . F . . . . . . . . V . . . . . . . . A . L A . . . . . . . . . . . . . . . . . . . . . K K L N . PN P . RT H . . RT A P . K RNGR . A . . . . A P . . . L . I N . . . . . S . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . V . . . . . . . . A . . A . . . . . . . . . . . . . . . . I . . . . . K L N . . S . . . A . . . S . A . . I RN SR . . A . . . A PH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P . . . . . . . . . A R . . . F . . . L . . S . . . . . S . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . A . . . . . A . . I . . . R . . RGE . T P . G . . . A . . S . . . R . . . . I . . . . . . . . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . I . . . . . . . . . . . . . P . P S . S . Y - - - - - - - - PANH L . . . . . . A F . . . . . A A . . . . . . . . . . . . . . V . . . . . . . . Determination of virulence . . . . . F . . . . . . . . . . . . . . . L . AN . . . . . . . . . . . . . . . . . . . . . . . . . . P . P S . . WH - - - - - - - - PA . H L . . . R . . A F . . . . Q . E . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PG . . R P . . . . V . . . P . . . . . A . . . . . F . N . . . T T T GA CAGT CAGGT GA A T GGCCGCGA T T GGCGT GT GGCCT CT GAGT CA CCT A T T CA A T T AGGGCGA T CA CA T GGGGGT CA T A CT T A A T CAGGCAGGA A CCA T GT GA CCGA A A T T . . . . GT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . T . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . A . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . G . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . T . . . . . A . . . . . . . . . . . . . . . . . . . . . . . G . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A B C D Figure 3 Multiple alignments of partial nsp2 (A), GP3 (B) and GP4 (C) amino-acid sequences of eleven different European PRRSV strains. Only aa differing from Lelystad virus (LV) are shown, with identical aa represented by dots. Deletions compared to LV are marked in red. Deletions of AUT14-440 are highlighted with solid boxes; a deletion in all three isolates is indicated with a dotted box. The numbers above the alignment indicate the position in the protein. A A single aa deletion is present in all three isolates and KNU-07 at position 182 or 183 in nsp2. In AUT14-440 aa 320–323, aa 359–364 and aa 699–700 are deleted compared to LV. B, C The box marks a 12 aa deletion in the overlapping region of GP3 and GP4 of AUT14-440. D Multiple alignment of 3′-UTR nucleotide sequences of ten European PRRSV strains. Nucleotides differing from Lelystad virus (LV) are shown, with identica nucleotides represented by dots. A deletion of 1 nt at position 87 in AUT14-440 is marked in red and highlighted with a solid box. MA A A T L F F L AGAQH I MV SEA FA CK PC F ST H L SD I ET N T T A A AG FMV L QD I N C FR PHGV SA AQEK I S FGK S SQCR EA VGT PQY I T I T AN V T D E SY L YN AD L LM L SA C L F YA . . T . I . . L . V . . . C L . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . K . E . L Q . R . D . T T P SGV . YA . Q . . . . Q . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . I . L L . V . . . . L . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . K . E . L Q . - - - - - - - - - - - - K V R . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . L . V . . . R L V . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . K . E . SQ . . KN . ST SHGA PQT . R . . . . Q . I . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . I . . L . V . . . R L . . . . . . . . . . . . . . . . . . . K . . . . P . . N . . . . . K . E . L Q . . RG . T T . Q SV P YK . Q . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . Determination of virulence . E . P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . I . . L . . . . . Y L . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . L . . . . . . T . . . T . . . . . P . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . LMV . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . G . . N . D . L . H . R . T . . . GR . YT RGP . . . . . V . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . L . V . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . L . . . . . T . AQ S L R . . - - - - - - - - R . P P . . . . . . . I . . . . . . M . . . . . . . . . . . . . . . . . . . . . . . . . V . . I . . L . V S . . Y L V . . . . . . . . . . . . . Determination of virulence . . . N . K . . . . . . . . . L . . . . . T . AQ S . V A . - - - - - - - - S . P . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A F . L . V . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . N . . . S . . . EA . . T . GQV P SR . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T GHN I SA L YA A Y YHHQ I DGGNWFH L EWL R P L F S SWL V L N I SWF L RR SPV SPV SRR I YQ I L R PT R PR L PV SWS FRT S I V SD L T G SQQRK RK F P SE SR PN V V K P SV L P ST SR . N Y . . . . S . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . V . . . . . . . . A . . A . . . . . . . . . . . . . . . . . . . . . K K L N . FN . A . T . . . R . A . . MRN SR . . A . L . A . . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . Determination of virulence . . L . A . . A . . . . . . . . . . . . . . . . . . . . . . K L N A . N P - - - - - - - - - - - - R YGH S . . . . . . . . . . L Q VN . . . . . S . . . . . . . . . . . . . . . . . . . . . . F . . . . . . . . V . . . . . . . . A . L A . . . . . . . . . . . . . . . . . . . . . K K L N . PN P . RT H . . RT A P . K RNGR . A . . . . A P . . . L . I N . . . . . S . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . V . . . . . . . . A . . A . . . . . . . . . . . . . . . . I . . . . . K L N . . S . . . A . . . S . A . . I RN SR . . A . . . A PH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . Determination of virulence . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P . . . . . . . . . A R . . . F . . . L . . S . . . . . S . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . A . . . . . A . . I . . . R . . RGE . T P . G . . . A . . S . . . R . . . . I . . . . . . . . . V . . . . . . . . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . I . . . . . . . . . . . . . P . P S . S . Y - - - - - - - - PANH L . . . . . . A F . . . . . A A . . . . . . . . . . . . . . V . . . . . . . . . . . . . F . . . . . . . . . . . . . . . L . AN . . . . . . . . . . . . . . . . . . . . . . . . . . P . P S . . Determination of virulence WH - - - - - - - - PA . H L . . . R . . A F . . . . Q . E . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PG . . R P . . . . V . . . P . . . . . A . . . . . F . N . . . T T T GA CAGT CAGGT GA A T GGCCGCGA T T GGCGT GT GGCCT CT GAGT CA CCT A T T CA A T T AGGGCGA T CA CA T GGGGGT CA T A CT T A A T CAGGCAGGA A CCA T GT GA CCGA A A T T . . . . GT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . T . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . T . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . G . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . Determination of virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . T . . . . . A . . . . . . . . . . . . . . . . . . . . . . . G . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of virulence . . . . . . . . . . . B C D Figure 3 Multiple alignments of partial nsp2 (A), GP3 (B) and GP4 (C) amino-acid sequences of eleven different European PRRSV strains. Only aa differing from Lelystad virus (LV) are shown, with identical aa represented by dots. Deletions compared to LV are marked in red. Deletions of AUT14-440 are highlighted with solid boxes; a deletion in all three isolates is indicated with a dotted box. The numbers above the alignment indicate the position in the protein. A A single aa deletion is present in all three isolates and KNU-07 at position 182 or 183 in nsp2. In AUT14-440 aa 320–323, aa 359–364 and aa 699–700 are deleted compared to LV. B, C The box marks a 12 aa deletion in the overlapping region of GP3 and GP4 of AUT14-440. D Multiple alignment of 3′-UTR nucleotide sequences of ten European PRRSV strains. Nucleotides differing from Lelystad virus (LV) are shown, with identical nucleotides represented by dots. A deletion of 1 nt at position 87 in AUT14-440 is marked in red and highlighted with a solid box. B Figure 3 Multiple alignments of partial nsp2 (A), GP3 (B) and GP4 (C) amino-acid sequences of eleven different European PRRSV strains. Only aa differing from Lelystad virus (LV) are shown, with identical aa represented by dots. Deletions compared to LV are marked in red. Deletions of AUT14-440 are highlighted with solid boxes; a deletion in all three isolates is indicated with a dotted box. The numbers above the alignment indicate the position in the protein. A A single aa deletion is present in all three isolates and KNU-07 at position 182 or 183 in nsp2. In AUT14-440 aa 320–323, aa 359–364 and aa 699–700 are deleted compared to LV. B, C The box marks a 12 aa deletion in the overlapping region of GP3 and GP4 of AUT14-440. D Multiple alignment of 3′-UTR nucleotide sequences of ten European PRRSV strains. Nucleotides differing from Lelystad virus (LV) are shown, with identical nucleotides represented by dots. A deletion of 1 nt at position 87 in AUT14-440 is marked in red and highlighted with a solid box. Figure 3 Multiple alignments of partial nsp2 (A), GP3 (B) and GP4 (C) amino-acid sequences of eleven different European PRRSV strains. Only aa differing from Lelystad virus (LV) are shown, with identical aa represented by dots. groups (p = 0.055 between GER09-613 and AUT14-440 for the third week). groups (p = 0.055 between GER09-613 and AUT14-440 for the third week). occurrence in different lung lobes and on the severity of the lesions, a total score per lung was calculated, applying a PRRSV specific scoring system that has been described elsewhere [27]. The mean values for the Austrian iso- lates were 52.4 ± 20.9 for AUT14-440 and 53.6 ± 9.4 for AUT13-883 (Figure 5A). In these two groups all types of lesions mentioned above were present in mild to severe manifestations with no visible differences between the groups. The results differed significantly (p < 0.01) from both the control group with a mean score of 5.6 ± 3.0 and group GER09-613, which mainly showed minor accumu- lations of inflammatory cells (mean histological score of Determination of virulence Deletions compared to LV are marked in red. Deletions of AUT14-440 are highlighted with solid boxes; a deletion in all three isolates is indicated with a dotted box. The numbers above the alignment indicate the position in the protein. A A single aa deletion is present in all three isolates and KNU-07 at position 182 or 183 in nsp2. In AUT14-440 aa 320–323, aa 359–364 and aa 699–700 are deleted compared to LV. B, C The box marks a 12 aa deletion in the overlapping region of GP3 and GP4 of AUT14-440. D Multiple alignment of 3′-UTR nucleotide sequences of ten European PRRSV strains. Nucleotides differing from Lelystad virus (LV) are shown, with identical nucleotides represented by dots. A deletion of 1 nt at position 87 in AUT14-440 is marked in red and highlighted with a solid box. Page 8 of 13 Sinn et al. Vet Res (2016) 47:17 B A 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Figure 4 Clinical signs and daily weight gain. A Pigs were examined daily and a clinical score was calculated based on the severity of PRRSV- associated clinical signs. The mean values for each group are shown with a continuous line and single spots represent individual animals. As group AUT14-440 was euthanized on day 14 pi there are values for 1 day more than for the other groups. B Pigs were weighed at six time points and daily weight gain was calculated for three time frames: 1st week (−7 to 0 dpi), 2nd week (0 to 7 dpi) and 3rd week (7 to 13 dpi) of the trial. Whiskers represent maximum and minimum values. Differences between the groups were visible for the last time period, 7–13 dpi, although they are not statistically significant (p = 0.055 between GER09-613 and AUT14-440). A B 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd B Figure 4 Clinical signs and daily weight gain. A Pigs were examined daily and a clinical score was calculated based on the severity of PRRSV- associated clinical signs. The mean values for each group are shown with a continuous line and single spots represent individual animals. As group AUT14-440 was euthanized on day 14 pi there are values for 1 day more than for the other groups. Determination of virulence B Pigs were weighed at six time points and daily weight gain was calculated for three time frames: 1st week (−7 to 0 dpi), 2nd week (0 to 7 dpi) and 3rd week (7 to 13 dpi) of the trial. Whiskers represent maximum and minimum values. Differences between the groups were visible for the last time period, 7–13 dpi, although they are not statistically significant (p = 0.055 between GER09-613 and AUT14-440). Figure 4 Clinical signs and daily weight gain. A Pigs were examined daily and a clinical score was calculated based on the severity of PRRSV- associated clinical signs. The mean values for each group are shown with a continuous line and single spots represent individual animals. As group AUT14-440 was euthanized on day 14 pi there are values for 1 day more than for the other groups. B Pigs were weighed at six time points and daily weight gain was calculated for three time frames: 1st week (−7 to 0 dpi), 2nd week (0 to 7 dpi) and 3rd week (7 to 13 dpi) of the trial. Whiskers represent maximum and minimum values. Differences between the groups were visible for the last time period, 7–13 dpi, although they are not statistically significant (p = 0.055 between GER09-613 and AUT14-440). Viremia and virus shedding To exclude the possibility of cross-contamination between the groups, 50 µL of serum taken 3 dpi from one animal per group (#10, 14, 19) were inoculated onto naïve PAM and virus was re-isolated. RT-PCR and subse- quent sequencing of the 700 bp PCR amplicon covering ORF4 confirmed the identity of the viral strains. Com- pared to the parental strain, two point mutations were detected in each strain. One of them was the same for all three strains and resulted in an amino-acid change from aspartic acid to asparagine at position 33 in GP4. In addi- tion melting temperatures of amplicons from positive nasal swabs confirmed that no cross-contamination had occurred (data not shown). To examine virus shedding from the upper respiratory tract, nasal swabs were taken at −7, 3, 7, 10 and 13 dpi and analysed with qRT-PCR for PRRSV-specific nucleic acids (data not shown). All animals from infected groups were virus-positive 3 dpi. Group GER09-613 tested com- pletely negative on day 7 pi and one pig (#8) remained negative until the end of the trial, while the other animals tested positive on day 10 and 13 pi. Virus shedding could be determined for four pigs each from groups AUT14- 440 and AUT13-883 on study days 7 and 10, whereas only two animals from group AUT14-440 and all ani- mals from group AUT13-883 were positive on the last Viral nucleic acids in serum could be detected with qRT-PCR as early as day 3 pi in all animals from group AUT14-440 and in two animals from group AUT13- 883 (Figure 6). In the GER09-613 group, the first virus- positive animals were detected 10 dpi. On day 13 pi all animals from this group except one (#8) tested positive for viral RNA; animal #8 remained negative for qRT- PCR as well as for titration on PAM (data not shown) on all sampling days. The number of genomic copies in the serum of animals infected with AUT14-440 declined Figure 5 Histological lung lesions. For each animal, a histopathological lung lesion score was calculated by considering the degree of PRRSV- associated lesions in every lung lobe. Histological lesion scores are presented A in a table and B as a box-plot. Mean score (±standard deviation) and individual scores for each animal are displayed for all groups. B Whiskers represent maximum and minimum values. Gross pathology and histopathology At 13 and 14 dpi, pigs were euthanized and necropsied with special emphasis on the lungs. No other organs than lungs showed pathological lesions. Samples from each lung lobe were fixed in formalin, embedded in paraffin, stained with hematoxylin and eosin and examined his- tologically. Lesions were hypertrophy and hyperplasia of pneumocytes, septal infiltration by mononuclear cells, perivascular and intraalveolar accumulation of inflam- matory cells and necrotic debris. Depending on their Page 9 of 13 Sinn et al. Vet Res (2016) 47:17 15 ± 6.0). The scores for each group are displayed as a boxplot in Figure 5B. towards the end of the trial. In the AUT13-883 group, the onset of viremia differed between the pigs. The max- imum number of genome copies per mL serum was up to 100 times higher in the AUT14-440 group than in the other groups. On days 3 and 7 pi, the difference between group AUT14-440 and all other groups was significant (p < 0.01), whereas on day 10 pi only the difference to the control group and the AUT13-883 group was significant (p < 0.05). Serum titres on PAM were also highest in the AUT14-440 group (up to 2.3 × 103 TCID50/mL, data not shown). Viral RNA or infectious virus could be detected in the serum of all infected animals, except for #8, with qRT-PCR and titration on PAM on one or more sampling days. The animals from the control group gave negative results with both assays throughout the trial. Viremia and virus shedding Letters indicate significant differences (p < 0.01) from control group (a), group GER09-613 (b), group AUT14-440 (c) and group AUT13-883 (d). Figure 5 Histological lung lesions. For each animal, a histopathological lung lesion score was calculated by considering the degree of PRRSV- associated lesions in every lung lobe. Histological lesion scores are presented A in a table and B as a box-plot. Mean score (±standard deviation) and individual scores for each animal are displayed for all groups. B Whiskers represent maximum and minimum values. Letters indicate significant differences (p < 0.01) from control group (a), group GER09-613 (b), group AUT14-440 (c) and group AUT13-883 (d). Sinn et al. Vet Res (2016) 47:17 Page 10 of 13 g day, day 13. The nasal swabs from the control ested negative for PRRSV-specific nucleic acids at me point. y dy titres against PRRSV were determined at the ng of the trial to ensure the PRRSV-free status of mals. At this time point all pigs tested negative for fic antibodies with the commercial IDEXX X3 (Figure 7). Pigs were further screened for sero- ion 7, 10 and 13 dpi. First detection of PRRSV-i antibodies was possible 7 dpi in the sera of two m groups AUT14-440 and GER09-613. On days 13 pi all infected animals but one (#19) tested pos- ELISA. The clearest antibody response with the P/PK ratios was seen in the AUT14-440 group. mals from the control group were negative for i 6 Viral load in serum quantified by qRT-PCR. For each infected group qRT-PCR results are displayed as genome copies per mL serum 3, 7, 10 and 13 dpi. Whiskers represent maximum and minimum values. Letters indicate significant differences (p < 0.05) to group GER09-613 14-440 (b) and AUT13-883 (c). -7 7 10 13 -7 7 10 13 -7 7 10 13 Figure 7 Antibody response to PRRSV infection. ELISA IDEXX X3 results as a ratio of the optical density (OD) of the sample and the OD of the positive control for infected groups at −7, 7, 10 and 13 dpi. Values over the cut-off at 0.4 (dotted line) were regarded as positive. Whiskers represent maximum and minimum values. Figure 6 Viral load in serum quantified by qRT-PCR. For each infected group qRT-PCR results are displayed as genome copies per mL serum for −7, 3, 7, 10 and 13 dpi. Whiskers represent maximum and minimum values. Serology Antibody titres against PRRSV were determined at the beginning of the trial to ensure the PRRSV-free status of the animals. At this time point all pigs tested negative for N-specific antibodies with the commercial IDEXX X3 ELISA (Figure 7). Pigs were further screened for sero- conversion 7, 10 and 13 dpi. First detection of PRRSV- specific antibodies was possible 7 dpi in the sera of two pigs from groups AUT14-440 and GER09-613. On days 10 and 13 pi all infected animals but one (#19) tested pos- itive in ELISA. The clearest antibody response with the highest P/PK ratios was seen in the AUT14-440 group. The animals from the control group were negative for PRRSV-specific antibodies at every time point. Figure 7 Antibody response to PRRSV infection. ELISA IDEXX Figure 7 Antibody response to PRRSV infection. ELISA IDEXX X3 results as a ratio of the optical density (OD) of the sample and the OD of the positive control for infected groups at −7, 7, 10 and 13 dpi. Values over the cut-off at 0.4 (dotted line) were regarded as positive. Whiskers represent maximum and minimum values. Figure 7 Antibody response to PRRSV infection. ELISA IDEXX X3 results as a ratio of the optical density (OD) of the sample and the OD of the positive control for infected groups at −7, 7, 10 and 13 dpi. Values over the cut-off at 0.4 (dotted line) were regarded as positive. Whiskers represent maximum and minimum values. complete genome sequences of two recent Austrian field isolates but also to their characterization in vivo. (Phylo-) genetic information could be interpreted in the light of the phenotypic properties of the isolates. This is especially important because genetic relationships do not give evidence for the virulence of PRRSV strains [6]. To go beyond the descriptive determination of selected sequences, it is necessary to address the pathogen’s prop- erties by carefully characterizing the isolate. Viremia and virus shedding Letters indicate significant differences (p < 0.05) to group GER09-613 (a), AUT14-440 (b) and AUT13-883 (c). Figure 6 Viral load in serum quantified by qRT-PCR. For each infected group qRT-PCR results are displayed as genome copies per mL serum for −7, 3, 7, 10 and 13 dpi. Whiskers represent maximum and minimum values. Letters indicate significant differences (p < 0.05) to group GER09-613 (a), AUT14-440 (b) and AUT13-883 (c). sampling day, day 13. The nasal swabs from the control group tested negative for PRRSV-specific nucleic acids at every time point. -7 7 10 13 -7 7 10 13 -7 7 10 13 Figure 7 Antibody response to PRRSV infection. ELISA IDEXX X3 results as a ratio of the optical density (OD) of the sample and the OD of the positive control for infected groups at −7, 7, 10 and 13 dpi. Values over the cut-off at 0.4 (dotted line) were regarded as positive. Whiskers represent maximum and minimum values. -7 7 10 13 -7 7 10 13 -7 7 10 13 Discussion Austria is surrounded by seven neighbouring countries and lies on the border between Western and Eastern Europe. Due to its central location it has become a major European corridor for travel and the movement of goods, including live pigs. In fact, one of the isolates (AUT440- 14) originated from a farm directly adjacent to the major west-east transit road. The reintroduction of PRRSV into virus-free herds is the most urgent yet least understood problem for the swine industry in Austria. Epidemiologi- cal evidence is often insufficient as only few ORF5 and ORF7 sequences from Austria are accessible at GenBank, most of them originating from only three publications [15–17]. In none of these cases were sequence data cor- related to the biological properties of the isolates. The initial virus isolation in this study was performed on primary PAM, which are believed to be the main target cells of PRRSV in vivo and are widely used for PRRSV-1 isolation and propagation [28]. The much more convenient simian cell line MARC-145 can usually not be used for unadapted PRRSV-1 virus strains, although it is readily infected by PRRSV-2 without adaptation [6, 28]. Interestingly, it also supported growth of the first passage In this study, the initial question about the phyloge- netic background of two field strains not only led to Page 11 of 13 Sinn et al. Vet Res (2016) 47:17 Page 11 of 13 A closer look at the full genome sequences revealed additional deletions in strain AUT14-440. In nsp2, three more deletions of 2–6 aa support the conclusion from the phylogenetic analysis that the isolate is not as closely related as the three other strains. The 12 aa deletion in the overlapping region of GP3 and GP4 is unique to this strain. Other published isolates that carry a deletion in this highly variable area only miss 1–8 aa compared to LV and the concomitant occurrence of deletions in nsp2 and ORF3/4 has only been described for a few Chinese strains [31–33]. The first strains carrying a deletion in the overlapping region of ORF3 and ORF4 were isolated in Denmark as long ago as 1992, shortly after the discovery of PRRSV. In subsequent evaluations, deletion mutants were reported to evolve more quickly than non-deleted viruses [34]. Discussion This might be due to immunological pres- sure, since neutralizing antibodies against this region in GP4 have been described in vitro for LV [35] and two Belgian strains [36]. This region and the corresponding region in GP3 were also shown to be immunogenic for other isolates, including a Danish deletion mutant with an 8 aa deletion [34, 37]. Therefore, it is very likely that the highly variable region in the overlap of GP3 and GP4 is under negative selection pressure, causing deletions or mutations to occur [38]. This is in line with the existence of very similar aa sequences next to the deletion in the Austrian isolates and their nearest relatives GER09-613 and KNU-07, indicating that the four strains evolved in a similar direction, probably due to immunological pres- sure. The 12 aa deletion in AUT14-440 might prevent the binding of GP4-specific antibodies and thereby result in an evolutionary advantage. This idea will be tested in fur- ther experiments. of AUT14-440, a PRRSV-1 isolate. This observation encouraged us to investigate the phylogenetic classifica- tion of the strain. Virus isolation is rarely performed in routine PRRSV diagnostics and sequences are normally obtained directly from clinical samples. Generally, only ORF5 or ORF7 sequences are determined. However, there are doubts about the validity of phylogenetic trees based solely on ORF5 or ORF7 sequences because of the recombination potential and high rate of nucleotide substitution shown by these genes [6, 14]. As a matter of fact, in the phyloge- netic trees we determined based on ORF5 and 7 the rela- tionship between the two Austrian isolates and closely related strains remained unclear as AUT13-883 grouped differently in the two trees. The difference might relate to recombination events, which are believed to have a major impact on PRRSV diversity [6, 29]. No evidence for this phenomenon was found by recombination anal- ysis, probably due to the small number of full PRRSV-1 genomes available in GenBank (data not shown). This finding underlines the need for more full-length genome sequencing. The phylogenetic tree based on full genomes clearly showed relatedness between the two Austrian isolates, the German strain GER09-613 and the South Korean isolate KNU-07. Discussion However, it has to be considered that the four strains cluster together in a phylogenetic tree despite not being closely related (identities between 85.4 and 90.2%) and that only 38 PRRSV-1 strains were available for comparison, most of them from earlier than 2010. The picture might be less clear if the full genome sequences of more (recent) strains were available for inclusion in the analysis. At present we can only specu- late whether the South Korean strain was imported to Europe or whether the cluster is the result of convergent evolution. It is also possible that all four strains originate from older strains from Germany (e.g., BH_95_10-08_ EU) that show close relationships in ORF5 and ORF7 but for which complete sequences are not available. Detailed epidemiological statements will not be possible without a broader range of full-length PRRSV genomes. Another interesting aspect of the deletion is the fact that the heterotrimeric minor glycoproteins GP2, GP3 and GP4 are abundant on the viral surface and probably determine cell tropism [39]. A deletion of 12 aa in two of these proteins might lead to an altered cell tropism, as seen in this study for AUT14-440, which is, despite its type 1 genotype, capable of infecting MARC-145 cells. The single nt deletion in the 3′UTR of AUT14-440 has not been described in other PRRSV-1 strains, although there is a report of a deletion at a different position within the 3′UTR in highly pathogenic Chinese strains [40]. As the 3′UTR is essential for PRRSV replication [41], deletions in this area might have an implication for the replication efficacy of the virus. Nevertheless, comparing not only a small part of the 15 kb PRRSV genome but full sequences revealed remarkable characteristics of the strains and provided indications that the four strains have a common origin. AUT13-883 and KNU-07 have a proline deletion in nsp2 that has only been noted in several strains from Hong Kong [30]. AUT14-440 and GER09-613 also share a sin- gle aa deletion in nsp2, a single position after the proline. Additionally, on either side of the 12 aa deletion in GP3 in AUT14-440 the four strains share areas of aa sequences that are very similar to one another but clearly differ from those of other PRRSV-1 strains, providing further indications of a possible relationship. Discussion fi AUT14-440 differed from the other Austrian iso- late AUT13-883 and the German isolate GER09-613 also in other aspects. It was the only strain to cause clear respiratory distress in pigs in the animal trial. Animals infected with AUT14-440 ceased to increase daily weight gain in the second week after infection, Page 12 of 13 Sinn et al. Vet Res (2016) 47:17 highlighting the economic relevance of the strain. Although pigs from the other groups remained rela- tively healthy throughout the trial, moderate to severe lung lesions were found in pigs infected with AUT13- 883, which also showed varying developments of daily weight gain. It is conceivable that clinical signs would have been more prominent in all infection groups if a less natural route of infection, e.g., intramuscular, or higher viral titres had been chosen. constant evolutionary pressure, both from vaccination and from instruments of modern herd management, it is of the utmost importance to keep up with the increasing diversity of PRRSV by studying current isolates. Additional file 2. Phylogenetic analysis of ORF7. Additional file 2. Phylogenetic analysis of ORF7. Phylogenetic tree based on ORF7 nucleotide sequences of 41 PRRSV-1 strains and PRRSV-2 prototype VR2332 as an out-group as well as the two closest related sequences in NCBI BLASTn phylogenetic tree for each isolate. The PRRSV strains presented in this study are marked in red. The tree was constructed using the neighbour joining method with the numbers at the nodes representing bootstrap values in % of 1000 replicates. Scale bar: number of substitutions per site In the present study, high amounts of viral RNA in blood were associated with severity of clinical signs, with titres of AUT14-440 exceeding those of the other strains by up to 100-fold. In contrast, the amounts of viral RNA in nasal swabs were lowest for pigs infected with AUT14- 440, indicating that the amount of virus shedding via the respiratory route is not necessarily correlated with viremia. The detection of virus shedding 3 dpi is prob- ably an artefact of intranasal infection, as pigs from group GER09-613 do not show any viral RNA in nasal swabs 7 dpi, which corresponds to the late onset of viremia in this group. Authors’ contributions LJS, HK, BL, CR, IH-P and TR planned and conceived the experiments. LJS, LZ, HK and IH-P performed the animal trial. GM and BJ provided field samples and executed qRT-PCR on nasal swabs. LJS, LZ and BL performed all other labora‑ tory work and analysed the data. LJS, BL and TR interpreted the results and designed the figures. LJS and TR wrote the manuscript. All authors read and approved the manuscript. The early detection of non-neutralizing N-specific anti- bodies is in accordance with previous studies [42, 43]. The IDEXX X3 ELISA is not quantitative but the high P/ PK ratios of group AUT14-440 are in agreement with the high number of viral genome copies in the blood of ani- mals infected with this strain. Author details 1 1 Institute of Virology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria. 2 Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Vet‑ erinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria. 3 Animal Health Service Upper Austria, Molkereistraße 5, 4910 Ried im Innkreis, Austria. Received: 4 October 2015 Accepted: 10 December 2015 Received: 4 October 2015 Accepted: 10 December 2015 Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Acknowledgements The authors thank Herbert Weißenböck for careful examination of histological lung samples, Graham Tebb for detailed correction of the manuscript text, Alexander Tichy for valuable help with the statistical analysis and Ludwig Haas for providing the isolate GER09-613. In summary, we have determined the complete genome sequence of two recent Austrian field isolates of PRRSV and interpret the nucleic acid sequences and the phylog- eny in the light of the strains’ in vivo characteristics. Both isolates cluster with a German field isolate from 2009 and the South Korean KNU-07 strain in whole genome phy- logenetic analysis. One of the field isolates, AUT14-440, caused clinical signs in an animal experiment, grew on MARC-145 cells and showed exceptional deletions. These well characterized isolates represent an excellent basis for further studies on the implications of the described molecular properties on virus entry, replication and path- ogenicity. It remains unclear whether the similarities to East Asian strains are unique to European strains because of the lack of full genome sequences of strains currently present in the field. As PRRSV strains are subject to 1. Neumann EJ, Kliebenstein JB, Johnson CD, Mabry JW, Bush EJ, Seitzinger AH, Green AL, Zimmerman JJ (2005) Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 227:385–392 2. Nieuwenhuis N, Duinhof TF, van Nes A (2012) Economic analysis of outbreaks of porcine reproductive and respiratory syndrome virus in nine sow herds. Vet Rec 170:225 3. 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https://openalex.org/W2509371953	https://europepmc.org/articles/pmc5055194?pdf=render	English	null	Assessment of sites of marrow and extramedullary hematopoiesis by hybrid imaging in primary myelofibrosis patients	Cancer medicine	2,016	cc-by	3,738	© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 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. Cancer Medicine 2016; 5(9):2378–2384 Cancer Medicine 2016; 5(9):2378–2384 doi: 10.1002/cam4.835 doi: 10.1002/cam4.835 doi: 10.1002/cam4.835 Mario Ojeda-Uribe1, Olivier Morel2, Constantin Ungureanu2, Christophe Desterke3,4, Marie-Caroline Le Bousse-Kerdilès4,5 & Hatem Boulahdour2,6 Mario Ojeda-Uribe1, Olivier Morel2, Constantin Ungureanu2, Christophe Desterke3,4, Marie-Caroline Le Bousse-Kerdilès4,5 & Hatem Boulahdour2,6 1Service d’Hématologie Clinique et Unité de Thérapie Cellulaire, Hôpital E Muller, GHR Mulhouse Sud-Alsace, Mulhous 2Pôle d’Imagerie, CHU Besançon, France 3INSERM UMS33, Hôpital Paul-Brousse, Villejuif, France 4Université Paris Sud 11, Villejuif, France 5INSERM U1197, Hôpital Paul-Brousse, Villejuif, France 6EA 4662-Nanomedicine Lab, Imagery and Therapeutics, Hôpital J Minjoz, CHU Besançon, France 1Service d’Hématologie Clinique et Unité de Thérapie Cellulaire, Hôpital E Muller, GHR Mulhouse Sud-Alsace, Mulhouse, Fran 2Pôle d’Imagerie, CHU Besançon, France 3INSERM UMS33, Hôpital Paul-Brousse, Villejuif, France 4Université Paris Sud 11, Villejuif, France 5INSERM U1197, Hôpital Paul-Brousse, Villejuif, France 6EA 4662-Nanomedicine Lab, Imagery and Therapeutics, Hôpital J Minjoz, CHU Besançon, France 1Service d’Hématologie Clinique et Unité de Thérapie Cellulaire, Hôpital E Muller, GHR Mulhouse Sud-Alsace, Mulhouse, France 2Pôle d’Imagerie, CHU Besançon, France 3INSERM UMS33, Hôpital Paul-Brousse, Villejuif, France 4Université Paris Sud 11, Villejuif, France 5INSERM U1197, Hôpital Paul-Brousse, Villejuif, France 6EA 4662-Nanomedicine Lab, Imagery and Therapeutics, Hôpital J Minjoz, CHU Besançon, France Correspondence Mario Ojeda-Uribe, Service d’Hématologie Clinique et Unité de Thérapie Cellulaire, GHRMSA, Hôpital E Muller, 20 avenue du Dr Laennec, 68070 Mulhouse, France. Tel: +33(3) 89647755; Fax: +33(3) 89647786; E-mail: ojeda-uribem@ghrmsa.fr Keywords Extramedullary hematopoiesis, hybrid imaging, primary myelofibrosis, radionuclides, SPECT, spleen We investigated noninvasive procedures by hybrid imaging to assess the sites of active or inactive hematopoiesis in patients with primary myelofibrosis (PMF). To this end, we used two radionuclides, technetium 99m (99mTc) and indium 111-chloride (111In-Cl3), coupled with single-photon emission tomography/com- puted tomography (SPECT/CT). We studied five patients with PMF and one with secondary myelofibrosis (MF). The classical pattern of lower fixation of both tracers at the axial skeleton where the myelofibrotic process occurs and the reactivation of sites of active hematopoiesis at the distal skeleton were con- firmed. Coupling both radionuclides to SPECT/CT imaging allowed for more precise visualization of the sites of extramedullary hematopoiesis as those observed in the spleen and liver. Splenic high uptake of 111In-Cl3 coupled with SPECT/CT represents a pathognomonic feature of PMF. We conclude that, the hybrid imaging procedures that we studied might constitute an alternative noninvasive method for the screening of the whole-body marrow and, by this way, to assess the impact of targeted therapies in PMF patients in whom it is well known that the distribution of the hematopoietic active areas is disturbed. Hybrid imaging could also be useful for diagnostic purposes in cases of early PMF or in suspected cases of myelofibrosis secondary to polycythemia vera or essential thrombocythemia. Funding Information No funding information is provided. Received: 5 March 2016; Revised: 26 June 2016; Accepted: 1 July 2016 Received: 5 March 2016; Revised: 26 June 2016; Accepted: 1 July 2016 Cancer Medicine Open Access ORIGINAL RESEARCH Introduction bone is disrupted in myeloproliferative neoplasms, such as primary myelofibrosis (PMF) or secondary MF (sMF) [3, 4]. Signal transduction abnormalities in PMF-HSCs can be driven by somatic mutations, such as those involv- ing the genes JAK2, CALR, or MPL [5]. The dynamic relationship between the skeleton and bone marrow (BM) hematopoietic niches throughout life implies that clinical entities may arise from hematopoietic stem cells (HSCs) and/or BM microenvironment abnormalities [1, 2]. Two HSC niches are described: the endosteal/ osteoblastic niche, where HSCs interact with osteoblasts, and the perivascular niches, where HSCs are close to endothelial cells, mesenchymal stromal cells, and pericytes surrounding the BM vascular structures [1, 2]. The rela- tionship between HSCs, their niches, and the trabecular Primary myelofibrosis is associated with extramedullary hematopoiesis (EMH), usually with splenomegaly and occasionally, hepatomegaly. Several tracers are used in nuclear medicine to assess EMH and BM hematopoietic activity. Colloids labeled with 99mTc show the reticuloen- dothelial system [4]. After intravenous (IV) injection, 99mTc nanocolloids are rapidly cleared from the plasma. 2378 M. Ojeda-Uribe et al. Hybrid Imaging in Primary Myelofibrosis Table 1. Baseline clinical and biological characteristics of the patients studied and summary of the hybrid imaging patterns observed with the two radiopharmaceuticals used. Axial skeleton Spleen Liver Distal skeleton EMH SPECT/CT Disease status and molecular profile 99mTc 111In 99mTc 111In 99mTc 111In 99mTc 111In 99mTc 111In P1 Fixation Lower fixation Hyperfixation Hyperfixation Hyperfixation Fixation Fixation Higher fixation Neg Neg PMF-F V617F-JAK2pos P2 Fixation Fixation Fixation Hyperfixation Fixation Fixation Fixation Higher fixation Neg Neg PMF-F JAK2pos (exon12) CALRneg MPLneg P3 Lower fixation Lower fixation Fixation Hyperfixation Fixation Fixation Fixation Higher fixation Neg Neg PMF-F JAK2pos (exon12) CALRneg MPLneg P4 Lower fixation Fixation Fixation Hyperfixation Fixation Fixation Fixation Higher fixation Neg Neg PMF-F V671F- JAK2pos P5 Lower fixation Fixation Fixation Fixation Fixation Fixation No fixation No fixation Neg Neg Post-TE-MF V617F-JAK2pos P6 Very low fixation Fixation Fixation Hyperfixation Fixation Fixation Low fixation Higher fixation Neg Neg PMF-F V617F-JAK2pos SPECT/CT, single-photon emission tomography/computed tomography; EMH, extramedullary hematopoiesis; PMF, primary myelofibrosis. The physiological uptake in normal adult humans is as follows: liver, 70%; spleen, 10%; and BM, 15–20%. 111In- Cl3-transferrin scintigraphy reflects the erythropoietic activity of BM and other sites of active hematopoiesis. Following IV injection, it is rapidly coupled with trans- ferrin and cleared from the plasma. The physiological uptake is as follows: liver, 20%; spleen, 1%; BM, 30%; and kidney, 7%. Introduction Both radiopharmaceuticals show a physi- ological uptake in the axial skeleton, but not in the distal skeleton [6]. Cell imaging using single-photon emission tomography (SPECT) utilizes gamma-emitting radiopharmaceuticals to allow two-dimensional (2D) imaging acquisition. After reconstruction, visualization of three-dimensional (3D) images in multiple planes is possible. It is often coupled to computed tomography (CT) (SPECT/CT) [6]. Few data exist regarding hematopoiesis assessment in PMF using hybrid imaging. We have previously shown its applicability in the EMH diagnosis [7] . In PMF, we have also used this technique for many years and because of recent developments in its molecular aspects, we con- sider this noninvasive procedure might allow us to assess the impact of novel targeted therapies [5]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Study design Table 1. Baseline clinical and biological characteristics of the patients studied and summary of the We retrospectively studied hybrid imaging procedures we had performed in five untreated PMF patients and in one patient with essential thrombocythemia (ET) who was developing secondary MF (Table 1). These diagnostic procedures were performed according to the Helsinki declaration and approved by the local pluridisciplinary Oncology-Hematology Committee. All patients gave their informed consent, after receiving detailed information of the benefits and risks of the procedure. A CT was added to the routine radionuclide procedures for PMF, which is already widely used in nuclear medi- cine. Here, it was performed at low dose using the X-ray tube of our hybrid device that was also employed for attenuation correction (AC). This correction helped us in the uptake assessment of deep soft structures, such as the spleen and liver. Diagnosis of PMF and ET was made according to WHO 2008 recommendations [8]. Myelofibrosis severity was based on BM biopsies assessed according to international recommendations. Interestingly, with respect to the molec- ular features of the PMF patients studied, two of them presented mutations in the exon 12 of JAK2 without a previous clinical or biological history of polycythemia vera and this for at least 10 years before the diagnosis of PMF (Table 1). 2379 M. Ojeda-Uribe et al. Hybrid Imaging in Primary Myelofibrosis Hybrid imaging procedures: two tracers were employed: (1) 99mTc-marked nanocolloid (Nanocis, IBA, Gif-sur- Yvette, France) scintigraphy. Whole-body planar and focused SPECT acquisitions were performed using a dual head gamma camera (Hawkeye, GE Healthcare, Fairfield, CT, USA). The whole-body acquisitions were made 1 and 6 h after the IV administration of 99mTc nanocolloids using the following acquisition parameters: a low energy collimator, a sweep speed of 12 cm/min for the whole body, and 300 sec for static images. SPECT/CT (SPECT: 60 projections, OSEM: 2 iterations, 10 subsets, Butterworth: 0.48/10, 128 × 128 matrix; CT: slice thickness: 10 mm, 140 KV, 40 mA, 256 × 256 matrix) was also performed, and multiplanar reconstructions were made. (2) 111In- Cl3-transferrin scintigraphy (Covidien, Mallinckrodt Medical, Petten, The Netherlands) with SPECT/CT (60 projections; OSEM: 2 iterations, 10 subsets; Butterworth: 0.48/10; 128 × 128 matrix; CT: slices thickness: 10 mm, 140 KV, 40 mA, 256 × 256 matrix). Study design Scintigraphy image acquisitions were made 48 and 72 h after IV administra- tion of 111In- Cl3, with the following acquisition parameters: low energy collimator, a sweep speed of 10 cm/min for the whole body, and 600 sec for static images. was concomitant to the high uptake of this radionuclide in the spleen, although some patients (e.g., P4) showed lower liver uptake intensity (Fig. 2). Axial, distal skeleton, and splenic uptake profiles in patients with advanced myelofibrosis Regarding the splenic high uptake of 111In-Cl3, it can be considered to be pathognomonic of PMF or sMF, because it reflects the EMH developed in this setting. In patients with advanced fibrotic process, this high splenic uptake was associated to a similar pattern of high 111In-Cl3 fixa- tion in the distal skeleton that also reflects the develop- ment of active hematopoiesis or at least a significant erythroblastic activity in these areas. Secondary early myelofibrosis uptake profile Interestingly, in the sMF (post-ET) patient (P5), there was no 111In-Cl3 hyperfixation at the distal skeleton. Conversely, we observed a significant splenic fixation with both radiopharmaceuticals and a low fixation with 99mTc- nanocolloids in the axial skeleton (Figs. 1 and 2). Axial, distal skeleton, and splenic uptake profiles We consider, the more advanced the process of marrow fibrosis and HSC externalization is, the stronger is the 111In-Cl3 splenic uptake. In conclusion, when associated with a lower fixation of this same radionuclide in the axial skeleton, the combined pattern of strong 111In-Cl3 splenic and distal skeletal uptake was highly suggestive and pathognomonic of PMF (Figs. 2 and 3). In PMF patients, we observed a lower fixation of both tracers 99mTc and 111In-Cl3 in the axial skeleton and, conversely, a higher fixation in the distal skeleton (Figs. 1 and 2; Table 1). Moreover, using hybrid imaging of 111In- Cl3 coupled to SPECT/CT allowed for a more detailed visualization of the extent and intensity of the EMH detected in the spleen of the PMF patients we studied (Fig. 3). In patients with advanced fibrotic process (Fig. 2, patients 1 to 4 and 6), this high splenic uptake was asso- ciated to a similar pattern of high 111In-Cl3 fixation in the distal skeleton. Some questions remain unsolved regarding the mecha- nisms involved in the distal skeleton recruitment or EMH reactivation in circumstances where these hematopoietic areas were progressively inactive since birth and replaced with adipose tissue [9]. In adults over 25 years old, almost all the active BM (red marrow) is located in the axial skeleton and the nonactive BM (yellow marrow) is mostly located on the distal skeleton [10, 11]. Yellow marrow is composed approximately of 95% fat cells whereas red marrow composition is 40% fat cells and 60% hemat- opoietic cells [11]. Is the distal skeleton reactivation observed in PMF a consequence of HSCs that cannot seed in the physiological homing hematopoietic areas, which participate essentially in the axial skeleton in adults? Or is the fruit of HSC reactivation (and their microen- vironment) already present in the distal skeleton but simply dormant? In this latter case, we do not know whether HSCs carry the same molecular abnormalities than those of the malignant PMF clone. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Hepatic uptake profile No other clear EMH localization besides the spleen was observed, except at the hepatic level. The interpretation of liver fixation was uneasy because both radiopharma- ceuticals 99mTc nanocolloids and 111In-Cl3 show significant physiological uptake although more importantly in the case of 99mTc nanocolloids. Regarding this last radionu- clide, we observed higher hepatic fixation with 99mTc- nanocolloids in only one patient (P1) (Fig. 1). When we looked at 111In-Cl3 distribution and intensity patterns, we observed a high liver uptake in most patients and this © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 2380 Hybrid Imaging in Primary Myelofibrosis M. Ojeda-Uribe et al. Figure 1. 99mTc-nanocolloid (Nanocis, Iba) scintigraphy coupled to single-photon emission tomography/computed tomography acquisitions. Representative imaging of the results is shown here: UPN1 to UPN4 and UPN6 were primary myelofibrosis patients; UPN5 was a secondary (post-ET) MF patient. Figure 1. 99mTc-nanocolloid (Nanocis, Iba) scintigraphy coupled to single-photon emission tomography/computed tomography acquisitions. Representative imaging of the results is shown here: UPN1 to UPN4 and UPN6 were primary myelofibrosis patients; UPN5 was a secondary (post-ET) MF patient. MF patient. Interest in hybrid imaging The radiopharmaceutical patterns we observed in PMF have already been described, although without SPECT/ CT [12]. Hybrid imaging using these two radionuclides and SPECT/CT is a noninvasive procedure, allowing a larger and more precise visualization of the active/inactive hematopoietic areas in PMF. Moreover, regarding deep regions or overweight patients, AC can show more uptake The hybrid imaging pattern observed here evocates a shift of the active physiologic hematopoietic sites to other nonphysiologic sites following a sequence of hematopoietic reactivation starting in the spleen, followed by distal skel- eton recruitment and associated with progressive hemat- opoietic regression in the axial skeleton (Figs. 1 and 2). 2381 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Hybrid Imaging in Primary Myelofibrosis M. Ojeda-Uribe et al. Figure 2. 111In-Cl3-scintigraphy (Covidien) with single-photon emission tomography/computed tomography acquisitions. Representative imaging of the results is shown here. UPN1 to UPN4 and UPN6 were primary myelofibrosis patients; UPN5 was a secondary (post-ET) MF patient. focus potentially hidden because of soft tissue interposi-li Figure 2. 111In-Cl3-scintigraphy (Covidien) with single-photon emission tomography/computed tomography acquisitions. Representative imaging of the results is shown here. UPN1 to UPN4 and UPN6 were primary myelofibrosis patients; UPN5 was a secondary (post-ET) MF patient. Figure 2. 111In-Cl3-scintigraphy (Covidien) with single-photon emission tomography/computed tomography acquisitions. Representative imaging of the results is shown here. UPN1 to UPN4 and UPN6 were primary myelofibrosis patients; UPN5 was a secondary (post-ET) MF patient. focus, potentially hidden because of soft tissue interposi- tion between the emitting source and detector. This can represent an alternative to BM biopsies that are limited in their investigation to the iliac crest and do not allow a whole-body marrow screening and are also limited because of the possibility of sampling error. BM biopsies may well reflect the axial skeleton and the significant hematopoietic activity that occurs in this area in adults but they do not provide any information on distal skeleton or the liver and spleen hematopoiesis. Inflammatory versus myelofibrotic component of PMF Recent studies have highlighted the interest in other non- invasive procedures, such as 18F-fluorodeoxyglucose (18F- FDG) positron emission tomography/computed tomography (PET/CT), to assess the inflammatory com- ponent of PMF, which is a critical player in the PMF pathophysiology [13, 14]. Actually, 18F-FDG PET/CT in this setting might be considered a necessary complement © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 2382 Hybrid Imaging in Primary Myelofibrosis M. Ojeda-Uribe et al. Figure 3. 111In-Cl3 single-photon emission tomography/computed tomography imaging in one patient with very advanced primary myelofibrosis: (A) front view and (B) side view showing very intense uptake of the radiotracer in the spleen (yellow arrow) and almost no fixation in the backbone and other structures of the axial skeleton. A B B A component of PMF from 99mTc nanocolloids/SPECT/CT imaging, which reflects, in part, the activity of several cell types involved in inflammation. In conclusion, on the basis of the data from our study, we observed a cor- related time-dependent pattern of the shift of active sites of hematopoiesis detected by 111In-Cl3/SPECT/CT. B B A A Interestingly, the imaging patterns we observed with 111In-Cl3/SPECT/CT are quite similar to those observed with the use of another radiotracer, 3′-18Fluoro-3′-deox y-L-thymidine (18F-FLT) [15]. Moreover, in a few cases, hybrid imaging has been reported to be useful to detect some cases of EMH [7, 16]. Figure 3. 111In-Cl3 single-photon emission tomography/computed tomography imaging in one patient with very advanced primary myelofibrosis: (A) front view and (B) side view showing very intense uptake of the radiotracer in the spleen (yellow arrow) and almost no fixation in the backbone and other structures of the axial skeleton. In summary, and based on our preliminary results, we believe the use of hybrid imaging allowed (1) good evalu- ation of the extent and intensity of the sites of active/ inactive hematopoiesis in PMF patients; (2) this noninvasive procedure can also be useful in challenging differential diagnosis cases of NPMs or (3) when a myelofibrotic transformation of ET or polycythemia vera is suspected. However, the interest of this technique to assess the impact of targeted therapies needs further evaluation. of the hybrid imaging techniques we studied. However, the imaging patterns in PMF of the respective radiop- harmaceutical distribution are not similar. Inflammatory versus myelofibrotic component of PMF In a recent study, 18F-FDG PET/CT uptake was increased in the axial and distal skeleton when the degree of marrow fibrosis was low (grade I) and was rather mild when the degree of marrow fibrosis was high (grade III) [13]. The authors concluded a significant inverse correlation between the extent of the metabolically active disease (axial skeleton and distal skeleton plus spleen) and time since the diag- nosis of myelofibrosis [13]. This reported inverse correla- tion pattern with 18F-FDG PET/CT assessment contrasts with the 111In-Cl3/SPECT/CT imaging we observed in the sMF patient (P5) of our series who was at an early phase of the disease and showing a lower fixation to 99mTc nanocolloids and 111In-Cl3 at the distal skeleton. These data in early phases of myelofibrosis, can reflect a high inflammatory activity without achieving a reconversion of inactive into active BM at the distal skeleton. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Conflicts of Interest The authors have no conflicts of interest to disclose. References 1. Morrison, S. J., and D. T. Scadden. 2014. The bone marrow niche for haematopoietic stem cells. Nature 505:327–334. 2. Birbrair, A., and P. S. Frenette. 2016. Niche heterogeneity in the bone marrow. Ann. N. Y. Acad. Sci. 1370:82–96. doi: 10.1111/nyas.13016. [Epub ahead of print]. 3. Lataillade, J. J., O. Pierre-Louis, H. C. Hasselbalch, G. Uzan, C. Jasmin, M. C. Martyré, et al. 2008. Does primary myelofibrosis involve a defective stem cell niche? From concept to evidence. Blood 112:3026–3035. On the contrary, in patients with more advanced PMF (P1 to P4 and P6, Figs. 1 and 2) high fixation patterns of the distal skeleton were observed when both techniques were compared, implying that hematopoiesis reactivation at this level is also associated with an inflammatory process [13]. Moreover, age-related changes probably need to be considered if we want to assess the impor- tance of 18F-FDG PET/CT uptake values in patients with PMF [9]. 4. Martinaud, C., C. Desterke, J. Konopacki, L. Pieri, F. Torossian, R. Golub, et al. 2015. Osteogenic potential of mesenchymal stromal cells contributes to primary myelofibrosis. Cancer Res. 75:4753–4765. 5. Vannucchi, A. M., T. L. Lasho, P. Guglielmelli, F. Biamonte, A. Pardanani, A. Pereira, et al. 2013. Mutations and prognosis in primary myelofibrosis. Leukemia 27:1861–1869. In our series, most of the patients presented significant marrow fibrosis (grade III) and, as we already mentioned, these patients with advanced disease had 111In-Cl3 imaging patterns in favor of hematopoietic reactivation of the distal skeleton. Based on our observations, the splenic EMH that was well detected by 111In-Cl3/SPECT/CT appears to be associated with a high inflammatory process (Fig. 3). We can obtain some evidence of this inflammatory 6. Agool, A., A. W. J. M. Glaudemans, H. H. Boersma, R. A. Dierckx, E. Vellenga, and R. H. Slart. 2011. Radionuclide imaging of bone marrow disorders. Eur. J. Nucl. Med. Mol. Imaging 38:166–178. J. Nucl. Med. Mol. Imaging 38:166–178. 7. Moulin, V., C. Ungureanu, M. Ojeda-Uribe, S. Thiebault, C. Porot, O. Angoue, et al. 2012. Intrathoracic extramedullary hematopoiesis: the 2383 Hybrid Imaging in Primary Myelofibrosis M. Ojeda-Uribe et al. advantages of hybrid imaging. Diagn. Interv. Imaging 93:897–902. 13. Derlin, T., H. Alchalby, P. Bannas, S. Veldhoen, I. Apostolova, I. Triviai, et al. 2015. Assessment of bone marrow inflammation in patients with myelofibrosis: an 18F-fluorodeoxyglucose PET/CT study. Eur. J. Nucl. Med. Mol. Imaging 42:696–705. 8. Vardiman, J. W., J. Thiele, D. A. Arber, R. D. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. References Brunning, M. J. Borowitz, A. Porwit, et al. 2009. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009:937–951. 14. Desterke, C., C. Martinaud, N. Ruzehaji, and M. C. Le-Bousse-Kerdiles. 2015. Inflammation as a keystone of bone marrow stroma alterations in primary myelofibrosis. Mediators Inflamm. 2015:415024. doi: 10.1155/2015/415024. Epub 2015 Nov 12. 9. Blebea, J. S., M. Houseni, D. A. Torigian, C. Fan, A. Mavi, Y. Zhuge, et al. 2007. Structural and functional imaging of normal bone marrow and evaluation of its age-related changes. Semin. Nucl. Med. 37:185–194. 15. Andreoli, A., L. Vercellino, M. J. Ouvrier, E. Barré, B. Cassinat, V. de Beco, et al. 2014. Final results of a phase 1 study of 18F-FLT positron emission tomography (PET)/computed tomography imaging in myelofibrosis (FLT-MF-2009 study). Blood 124:3195 (abstract). 10. Cristy, M. 1981. Active bone marrow distribution as a function of age in humans. Phys. Med. Biol. 26:389–400. 11. Harstock, R. J., E. B. Smith, and C. S. Petty. 1965. Normal variations with aging of the amount of hematopoietic tissue in bone marrow from the anterior iliac crest. A study made from 117 cases of sudden death examined by necropsy. Am. J. Clin. Pathol. 43:326–331. 16. Ali, S. Z., M. J. Clarke, A. Kannivelu, D. Chinchure, and S. Srinivasan. 2014. Extramedullary pulmonary hematopoiesis causing pulmonary hypertension and severe tricuspid regurgitation detected by technetium- 99m sulfur colloid bone marrow scan and single-photon emission computed tomography/CT. Korean J. Radiol. 15:376–380. 12. Rain, J. D., and Y. Najean. 1993. Bone marrow scintigraphy in myelofibrosis. Nouv. Rev. Fr. Hematol. 35:101–102. 2384 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
https://openalex.org/W3205175782	https://zenodo.org/record/5228379/files/Flow%20diagram%20and%20included%20articles.pdf	English	null	Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a systematic review	F1000Research	2,021	cc-by	3,694	Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review e 1 - Flow diagram of the identification, screening, eligibility and included articles Figure 1 - Flow diagram of the identification, screening, eligibility and included articles Records identified by searching in Scopus (n = 253) Additional records identified in other databases (n = 72) Records after removing duplicates (n = 261) Selected articles (n = 261) Articles excluded at the title and abstract level (n = 144) Full-text articles evaluated for the study (n=117) Full-text articles excluded for failing to meet the inclusion criteria (n = 88) Items included in the study (n=29) identification Screening Eligibility Included Additional records identified in other databases (n = 72) Records after removing duplicates (n = 261) Articles excluded at the title and abstract level (n = 144) Full-text articles excluded for failing to meet the inclusion criteria (n = 88) Full-text articles evaluated for the study (n=117) Items included in the study (n=29) Table 1 - Included articles that met the eligibility criteria Table 1 - Included articles that met the eligibility criteria No. AUTHORS ARTICLE TITLE TYPE OF STUDY / CHARACTERISTICS OF THE STUDIES 1 Ahn E, Lee J, Jeon Y- H, Choi S-W, Kim E Anti-diabetic effects of mulberry (Morus alba L.) branches and oxyresveratrol in streptozotocin- induced diabetic mice Original Research (OR): Morus alba ethanol extracts were administered orally once daily for 22 days in amounts of 0,5 or 1 g/kg of body weight to a diabetic mouse, induced with streptozotocin. (1) 2 Chan EW-C, Lye P-Y, Wong S-K Phytochemistry, pharmacology, and clinical trials of Morus alba Systematic Review (SR): Morus alba leaves possess biological activities: antioxidants, antidiabetic, antihyperlipidemic, antiobesity, glycosidase inhibitor, cardioprotective, antimicrobial, and cytotoxic. (2) 3 Díaz SM, Cazaña MY, Pérez HY, Valdivia ÁA, Prieto AM, Lugo MY Qualitative evaluation of secondary metabolites in extracts of Morus alba L. (Mulberry) varieties and hybrids OR: The fresh leaves collected from Morus alba were washed, disinfected, dried, and pulverized; then, the extracts were obtained with n-hexane, ethanol, and water, which were filtered and subjected to phytochemical identification tests. A toxicity evaluation of the aqueous extracts was carried out in rats to determine their safety. (3) 4 Ge Q, Cheng L, Tang M, Zhang S, Liu L, Gao L, et al. Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review Analysis of mulberry leaf components in the treatment of diabetes using network pharmacology OR: The study identified 202 compounds of the Morus alba leaf, using mass spectrophotometry and gas chromatography, 22 components with curative properties were identified on Diabetes Mellitus. (4) 5 Go EJ, Ryu BR, Yang SJ, Baek JS, Ryu SJ, Kim HB, et al. Anti-obesity Effect of the Flavonoid Rich Fraction from Mulberry Leaf Extract OR: Flavonoid-rich fractions were administered to rats C57BL/6 mice fed a high-fat diet and their development in obesity was investigated. (5) 6 He X, Fang J, Ruan Y, Wang X, Sun Y, Wu N, et al. Structures, bioactivities and future prospective of polysaccharides from Morus alba (white mulberry): A review SR: The leaves and fruits are rich in polysaccharides with promising activities: antidiabetic, immunomodulatory, anti-inflammatory, antioxidant, antiobesity, hepatoprotective, and renoprotective. (6) Information Classification: General Information Classification: General 7 Jeszka-Skowron M, Flaczyk E, Jeszka J, Krejpcio Z, Król E, Buchowski MS Mulberry leaf extract intake reduces hyperglycaemia in streptozotocin (STZ)-induced diabetic rats fed high-fat diet OR: A high-fat diet was applied for 4 weeks to Wistar rats to induce type 2 diabetes. The animals were subsequently treated with extracts of dried leaves of Morus alba and ethanol or acetone. (7) 8 Ji S, Zhu C, Gao S, Shao X, Chen X, Zhang H, et al. Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes OR: The main chemical components of the ethanol extract of Morus alba leaves were analyzed to determine their identification and quantification using the high- performance liquid chromatography (HPLC) technique. Rats were used in which T2DM was induced using a high-fat diet combined with streptozotocin; the MLE was administered by oral gavage. (8) 9 Jiao Y, Wang X, Jiang X, Kong F, Wang S, Yan C Antidiabetic effects of Morus alba fruit polysaccharides on high-fat diet- and streptozotocin-induced type 2 diabetes in rats OR: Rats were induced to have diabetes on a high-fat, low-dose streptozotocin diet. The animals were treated with two polysaccharide fractions from the Morus alba fruit (MFP50 and MFP90). Normal rats and diabetic rats treated with metformin were compared. Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review (9) 10 Kan J, Velliquette RA, Grann K, Burns CR, Scholten J, Tian F, et al A novel botanical formula prevents diabetes by improving insulin resistance OR: Tests were carried out in male Sprague Dawley rats, administering extracts of a formula composed of extracts of leaves of Morus alba, American ginseng, and Trigonella foenum-graecum, to determine their inhibitory capacity on the enzymes α-amylase and α- glucosidase, in a cell-free system. (10) 11 Kar A, Mukherjee PK, Sankarshan S, Bahadur S, Ahmmed SKM, Subrata P Possible herb-drug interaction of Morus alba L.- a potential anti- diabetic plant from Indian traditional medicine OR: An assay was carried out to quantify the active compounds of the Morus alba leaf extract, by the RPHPLC method. The inhibitory potential was deter- mined in pooled CYP450 as well as recombinant CYP450. (11) 12 Khyade VB Influence of Leaf Decoction of Mulberry, Morus alba (L.) on Streptozotocin Induced Diabetes in Brown Rat, Rattus norvegicus OR: A test was carried out to evaluate the effects of treatment with the product resulting from the decoction of Morus alba leaves, on specimens of Rattus norvegicus”, for which, 20 g / L was administered. One week before treatment, experimental diabetes was induced by applying streptozotocin. (12) 13 Król E, Jeszka- Skowron M, Krejpcio Z, Flaczyk E, Wójciak RW The Effects of Supplementary Mulberry Leaf (Morus alba) Extracts on the Trace Element Status (Fe, Zn and Cu) in Relation to Diabetes Management and Antioxidant Indices in Diabetic Rats OR: Male Wistar rats were induced with diabetes by placing them on a high-fat diet and administering streptozotocin. 38 rats divided into 5 experimental groups were used: (1) AIN-93M-fed healthy control group; (2) HF control group; (3) HF diabetic group; (4) HF + AE diabetic group (6 g / kg diet); and, (5) HF + EE diabetic group (diet 6 g / kg). (13) 14 Lim SH, Yu JS, Lee HS, Choi C-I, Kim KH Antidiabetic Flavonoids from Fruits of Morus alba Promoting Insulin- Stimulated Glucose Uptake via Akt and AMP-Activated Protein Kinase Activation in 3T3-L1 Adipocytes OR: A phytochemical analysis of the Morus alba ethanol extract was performed using high-performance liquid chromatography (HPLC). After purification, it was possible to isolate two main active principles: rutin and quercetin-3-O-β-d- glucoside (Q3G), to later apply them to 3T3-L1 adipocytes. (14) 15 Liu C, Xiang W, Yu Y, Shi ZQ, Huang XZ, Xu L. Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review Comparative analysis of 1- deoxynojirimycin contribution degree to α-glucosidase inhibitory activity and physiological distribution in Morus alba L OR: 12-day-old male Wanxi white geese were used, which were randomly as- signed to 4 treatment groups: (1) Control group with simple diet without 1-de- oxynojirimycin (DNJ); (2) Group L-DNJ; (3) Group M-DNJ; and, (4) HDNJ Group; these last 3 groups had complementary basic diets with DNJ: 0,05 mg / g, 0,1 mg / g, and 0,15 mg / g, respectively. The feeding lasted for 6 weeks. (15) 16 Mahboubi M Morus alba (mulberry), a natural potent compound in management of obesity SR: The objective of this review was to evaluate the potential effect of Morus alba as an antiobesity agent, therefore, various databases were searched: PubMed, Science Direct, Springer, Wiley, and Google), unpublished data (reports from R&D, thesis, and dissertations). (16) 17 Mahesh DS, Vidhathri BS, Vidyashree DN, Narayanaswamy TK, Subbarayappa CT, Muthuraju R Biochemical Composition and Pharmacological Properties of Mulberry (Morus spp.) - A Review SR: The biochemical composition and pharmacological properties of various mulberry species are reviewed. It includes its nutritional value, its phytochemical composition, its antioxidant activity, its mineral composition, its hypoglycemic activity, its antiobesity and hyperlipidemic action, its antioxidant, antidiabetic, anti-inflammatory, and antiallergic function, its vasoactive, neuroprotective, renoprotective, and anticancer action. (17) 18 Mellado-Orellana R, Salinas-Lezama E, Sánchez-Herrera D, Guajardo-Lozano J, Díaz-Greene EJ, Rodríguez-Weber FL Pharmacological treatment of diabe- tes mellitus type 2 directed to pa- tients with overweight and obesity SR: The evidence-based recommendations are oriented towards pharmacological and surgical intervention and changes in the lifestyle of obesity management as part of the comprehensive treatment of patients with type 2 diabetes mellitus. (18) 19 Meng Q, Qi X, Chao Y, Chen Q, Cheng P, Yu X, et al. IRS1/PI3K/AKT pathway signal involved in the regulation of glycolipid metabolic abnormalities by Mulberry (Morus alba L.) leaf extracts in 3T3-L1 adipocytes OR: The Morus alba leaf extract was prepared to contain the flavonoids (1 g / ml) and the routine of the flavonoids was determined using the high-performance liquid chromatography (HPLC) technique, with the Agilent 1260 system. (19) 20 Meng Q, Qi X, Fu Y, Chen Q, Cheng P, Yu X, et al. Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes OR: Skeletal cells were cultured in vitro, which were treated with or without flavonoids extracted from Morus alba. For in vivo studies, db /db mice were used for type 2 diabetes, with/without MLF therapy. Α-SMA immunofluorescence staining and Coomassie brilliant blue staining were used to identify differentiated L6 cells. The glucose level and the L6 ATP level were carried out by optical density detection and the viability of the cell was carried out by the MTT method. (20) 21 Rodrigues EL, Marcelino G, Silva GT, Figueiredo PS, Garcez WS, Corsino J, et al. Nutraceutical and Medicinal Potential of the Morus Species in Metabolic Dysfunctions SR: The genus Morus is used for the treatment and prevention of diabetes mellitus through its hypoglycemic action, as it has phenolic, anthocyanin, and flavonoid components, in greater or lesser concentration depending on the species, in the case of Morus alba, it has low concentrations of flavonoids and anthocyanins. (21) 22 Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N, et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International SR: 255 high-quality sources published between 1990 and 2018 from 138 countries were studied. Countries with low-quality data were extrapolated, based on geography, ethnicity, language, and economy. A regression was applied to generate estimates taking into account the specific age between 20-79 years and the prevalence of diabetes. (22) OR: A phytochemical analysis of the Morus alba ethanol extract was performed using high-performance liquid chromatography (HPLC). After purification, it was possible to isolate two main active principles: rutin and quercetin-3-O-β-d- glucoside (Q3G), to later apply them to 3T3-L1 adipocytes. (14) Information Classification: General Information Classification: General Diabetes Federation Diabetes Atlas, 9th edition 23 Seclén S Diabetes mellitus in Peru where we are going SR: Analyzes epidemiological data extracted from surveys carried out in Peru: ENDES, 2013; ENAHO, 2009-2010; PERUDIAB, 2012; WHO DIAMOND, SEARCH Study. (23) 24 Swathi P, Gana Manjusha K, Vivekanand M, Ramkishan A, Bhavani B Effect of morus alba against hyperglycemic and hyperlipidemic activities in streptozotocin induced diabetic nephropathy OR: It was applied in male Wistar rats, induced to diabetes with streptozotocin, applying an ethanolic extract of the leaf of Morus alba (AME). Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review After 12 weeks of starting the experimental work, the following were biochemically analyzed: blood glucose, glycosylated hemoglobin, bilirubin, albumin, creatinine, total protein, urea, lipid profile, and urine. Histopathological observations were also made. (24) 25 Villena JE Diabetes Mellitus in Peru SR: It was based on the systematic search of bibliographic sources included in Scielo, PubMed, literature from the World Health Organization, the International Diabetes Federation, and Peruvian agencies, related to the subject in question. (25) 26 Wei H, Liu S, Liao Y, Ma C, Wang D, Tong J, et al. A systematic review of the medici- nal potential of mulberry in treating diabetes mellitus SR: Morus alba studies are systematized, classifying them by their chemical composition, pharmacological effects of the different parts of the plant on diabetes mellitus, which included: glucose absorption, insulin secretion, oxidative and anti-inflammatory processes. (26) 27 Wen P, Hu TG, Linhardt RJ, Liao ST, Wu H, Zou YX A review of bioactive compounds and advanced processing technol- ogy SR: A description of mulberry is detailed examining its main active principles, which include polysaccharides, alkaloids, phenols, anthocyanins, and flavonoids. Technological advances for its extraction are described: solid-liquid extraction, supercritical-fluid extraction, liquid-pressurized extraction, microwave-assisted extraction, enzymatic assisted extraction, ultrasound- assisted extraction, solid-phase extraction; other techniques for its separation, such as ion-exchange chromatography, gel filtration chromatography, preparative liquid chromatography, countercurrent chromatography, silica gel chromatography, and macroporous resin adsorption. (27) 28 Wilson RD, Islam MS Effects of white mulberry (Morus alba) leaf tea investigated in a type 2 diabetes model of rats OR: It was carried out in male Sprague-Dawley rats of 6 weeks, divided into 4 groups; They were given high doses (0,5%) and low doses (0,25%) of white mulberry leaf tea. The groups were classified: normal control group (NC), dia- betic control group (DBC), high dose diabetic group (DMTH), and low dose di- abetic group (DMTL). T2DM was induced with streptozotocin and 4 weeks later analytical tests were performed to evaluate the levels of blood glucose, serum insulin, uric acid, glucose intolerance, glucose intolerance, fructosamine, AST, ALT, albumin, creatinine, and cholesterol. (28) 29 Younus I, Fatima A, Ali SM, Usmani S, Begum Z, Badar S, et al. Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a system- atic review A review of ethnobotany, phytochemistry, antiviral and cytotoxic / anticancer potential of morus alba linn SR: The article summarizes the phytochemistry, ethnobotany, antiviral and cytotoxic properties and anticancer potential of Morus alba Research revealed that various parts of the plant contained flavonoids, glycosides, polysaccharides, tannins, and lectins. (29) REFERENCES 1. Ahn E, Lee J, Jeon Y-H, Choi S-W, Kim E. Anti-diabetic effects of mulberry ( Morus alba L.) branches and oxyresveratrol in streptozotocin-induced diabetic mice. 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https://openalex.org/W4365449682	https://www.mrforum.com/wp-content/uploads/open_access/9781644902479/64.pdf	English	null	Investigation of the joining zone formation of impact extruded hybrid components by varied forming sequence and partial cooling	Materials research proceedings	2,023	cc-by	5,357	Content from this work may be used under the terms of the Creative Commons Attribution 3.0 license. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under license by Materials Research Forum LLC. 1Leibniz University Hannover, Institute of Forming Technology and Forming Machines, An der Universität 2, 30823 Garbsen, Germany 1Leibniz University Hannover, Institute of Forming Technology and Forming Machines, An der Universität 2, 30823 Garbsen, Germany apiwek@ifum.uni-hannover.de, bpeddinghaus@ifum.uni-hannover.de, apiwek@ifum.uni-hannover.de, bpeddinghaus@ifum.uni-hannover.de, p @ p g @ cuhe@ifum.uni-hannover.de, dbrunotte@ifum.uni-hannover.de, ebehrens@ifum.uni-hannover.de Keywords: Friction Welding, Hot Forging, Tailored Forming, Joining Zone, Partial Cooling Abstract. Hybrid material concepts enable the combination of beneficial properties of different materials to extend the limited potential of monolithic components. When it comes to steel and aluminium, a wear-resistant and a lightweight metal are combined to produce a weight-reduced high-performance component with load-adapted areas. A method to create hybrid gear shafts is a novel approach called Tailored Forming. The process chain consists of joining e. g. by friction welding and subsequent impact extrusion under elevated temperature. Before forming, an axial temperature gradient is set in the serial arranged semi-finished products to adjust the different yield stresses of the dissimilar materials through induction heating of the steel part. The subsequent forming is intended to positively influence the joining zone thermo-mechanically and geometrically. However, prior work indicated a limitation of the influence on the joining zone in forward rod extrusion. Therefore, approaches are being researched that enable a stronger formation of the joining zone geometry to influence the resulting bond qualities through surface enlargement. A forward rod extrusion process of friction welded hybrid semi-finished products made of 20MnCr5 (AISI 5120H) combined with EN AW-6082 (AA6082) was carried out experimentally. Complementary to prior investigations, in which mainly the aluminium section was reduced through the die angle followed by the steel, the forming sequence of the materials was reversed to increase the joining zone surface with variation of the forming path. Additionally, a cooling of the aluminium side was realized through an immersion cooling to adjust maximum temperature gradients and further equalize the different yield stresses. Hardness tests, metallographic and SEM images of cross-sections were taken to evaluate the bond quality with regard to the temperature influence, joining zone formation, occurring defects and the resulting intermetallic compound (IMC). Impact extrusion with initially steel formed followed by aluminium resulted in a spherical formation of the joining zone and consequently in greater surface area, but also lead to partial defects in the IMC. The partial cooling of the aluminium allowed higher temperature gradients to be set, thus reducing defects through improved material flow in the joining zone. Material Forming - ESAFORM 2023 Material Forming - ESAFORM 2023 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 State of the Art Within sheet metal forming, especially for the automotive industry, application-adapted hybrid semi-finished products called tailored blanks have been widely used since their development by ThyssenKrupp in 1983 [2]. Due to the local adaptations such as a local variation of the sheet thickness or sheet material, the components are optimised for a subsequent forming process or the end application [3]. This concept can also be applied to bulk metal forming. Contrary to compound forging of hybrid materials, where the joining is realised by forming [4-6], the hybrid semi-finished products are first pre-joined and then further processed by forming in order to positively influence the joining zone. The combination of joining and forming has been addressed by some studies for similar and dissimilar material pairings. For instance hot forging of cladded work pieces consisting of two different steel alloys using upsetting tests was investigated numerically and experimentally by Wang et al with regard to material distribution and the effect of friction [7]. Furthermore, in the studies by Foydl et al, an extrusion process was developed, in which aluminium profiles were reinforced by steel wires. For further processing into connecting rods, the components were cut to size and used as semi-finished products for a die forging process [8]. Basic research into joining by friction welding and subsequent upsetting of solid hybrid components made of copper and aluminium or steel and copper was carried out by Domblesky et al. The results obtained from upsetting indicate a good workability of hybrid materials [9]. Based on the concept of the studies mentioned, the new approach called Tailored Forming enables a production of load-adapted high-performance components by forming pre-joined hybrid semi-finished products. The process chain can differ in terms of joining processes used such as friction welding, lateral angular co-extrusion (LACE) or cladding and subsequent forming processes such as impact extrusion or die forging [10]. The joining zone of the two hybrid materials is usually the weakest area, where the failure of the component occurs. Therefore, the basic motivation is the enhancement of the weak point through suitable measures such as reducing tensile stresses and thus defects by a pressure superimposition [11-13], an adjustment of the yield stresses by a viable heating strategy [14] or improvement of the stress state by an inversed forming sequence [15]. Introduction An effective use of resources is becoming more crucial to deal with rising energy prices as well as increased environmental responsibilities. Monolithic components have their material-specific restrictions, thus new concepts and production techniques must be created to meet the rising standards. Monolithic materials cannot always meet the ever-more-complex and occasionally conflicting criteria put on lightweight constructions. One option to address these demands for lightweight concepts and attain a required load-adapted functionality is through hybrid components, which utilize different materials. In the case of steel and aluminium, the positive mechanical properties are combined to produce a high-performance component with low weight. 591 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Using the example of a hybrid gear shaft, aluminium can serve as a structural element, while steel is used in a power transmitting area [1]. State of the Art The last two aspects mentioned have a direct influence on the shape of the joining zone, which results in an increase of bond strength and quality through surface enlargement [15] and influencing IMCs, which in general are characterised by their brittleness. The creation of a sufficiently sound bond is the setting of the lowest possible IMC thickness, which should not exceed a certain thickness. According to Herbst et al. sufficiently narrow IMC thicknesses smaller than 1 μm indicate high bond strengths, while for thicknesses greater than 1 μm, the strength decreases significantly [16]. A sufficiently sound bound can be achieved through a subsequent forming operation. During the impact extrusion of serially arranged hybrid semi-finished products, it is crucial that the yield stress differences between the two materials are as small as possible. In this way, a high local degree of forming can be achieved and influence can be exerted on the joining zone geometry. If the yield stress difference between steel and aluminium is too high, the local deformation is not sufficient and the different materials flow one after the other into the extrusion shoulder with only a parallel displacement of the interface. In previous work [17], the influence on the joining zone properties after friction welding and impact extrusion under elevated temperatures of steel-aluminium billets was investigated. In various forming processes such as backwards cup (full forward) extrusion or hollow forward extrusion, it was possible to geometrically influence the joining zone so that larger joining zone surfaces were created. In the case of forward rod extrusion, however, the joining zone did not change and remained flat, almost as it was after friction welding (see Fig. 1). Since in forward rod extrusion no additional compressive stress is initiated by forming tools as in the other 592 592 Material Forming - ESAFORM 2023 Materials Research Forum LLC Materials Research Proceedings 28 (2023) 591-600 https://doi.org/10.21741/9781644902479-64 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 processes investigated, the axial temperature gradient is particularly important in the case in order to set the previously mentioned local deformations resulting in a spherical shape of the joining zone [17]. Due to this, a larger temperature gradient must be set in order to further equalise the yield stresses and also to influence the joining zone geometry. This is possible e. g. by actively cooling the aluminium. Fig. 1. Joining zone after friction welding and forward rod extrusion. Fig. 1. Joining zone after friction welding and forward rod extrusion. According to Gumm an increased flow rate of the centre material is observed if the harder metal undergoes the forming process first. In case the softer material is pressed through the die first, lower pressures in the forming zone and a formation of cavities in in the joining zone of compound forged components is observed [15]. Therefore, the sequence of materials is another crucial factor to further enhance the joining zone geometry through an improved stress state in the forming zone. Another aspect to reduce unwanted tensile stresses is the application of a pressure superimposition. Particularly in cold extrusion of hybrid materials, fractures occur inside the component due to differing yield stresses, which can be significantly reduced by this measure with the similar effect of the selection of material sequence [11-13]. However, this does not have any influence on the surface enlargement [17], but is required to ensure the reduction of defects. Deriving from the state of the art, a pressure-superimposed forward rod extrusion with partial aluminium-side cooling is carried out within the scope of this study by means of Tailored Forming. Furthermore, contrary to the previous work, the material sequence is inverted so that the steel is formed first followed by the softer aluminium. In order to reproduce the formation of the joining zone and the resulting surface enlargement, the forming path is varied. The two heating strategies (without vs. with cooling) are compared with regard to the joining zone formation. The process changes enable an improvement of the mentioned joining zone characteristics. 592 The friction welded and impact extruded steel-aluminium bonds are evaluated by means of metallographic images and hardness tests. Furthermore, the resulting IMC is characterised by SEM images and EDX analyses. The process changes enable an improvement of the joining zone characteristics. Tailored Forming for the Production of a Hybrid Transmission Shaft The process chain of Tailored Forming (see Fig. 2) first includes friction welding of the hybrid material pair consisting of steel (soft-annealed 20MnCr5) and aluminium (EN AW- 6082 T6 condition) in a serial arrangement. Then the joined semi-finished products are formed in a forward rod extrusion process with prior inductive heating, which in the context of this study takes place both with and without immersion cooling. This is followed by machining to produce the final component, which is characterised by a load-adapted material distribution. The process chain of Tailored Forming has already been used in earlier work to create customised, hybrid machine parts in serial and coaxial arrangements like axial bearing washers or bevel gears [10]. 593 593 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Materials Research Proceedings 28 (2023) 591-600 https://doi.org/10.2174 Materials Research Proceedings 28 (2023) 591-600 Fig. 2. Process chain of Tailored Forming for a hybrid shaft with reversed forming sequence. Rotatory Friction Welding Fig. 2. Process chain of Tailored Forming for a hybrid shaft with reversed form Fig. 2. Process chain of Tailored Forming for a hybrid shaft with reversed forming sequence. Rotatory Friction Welding Rotatory Friction Welding y g Before joining, the welding surfaces of both semi-finished products made of 20MnCr5 and EN AW-6082 are first machined and cleaned with ethanol to remove irregularities and impurities. After preparation, the materials are immediately joined on the KUKA Genius Plus friction welding machine with the experimental set-up and parameters shown in Fig. 3. a) b) Fig. 3. Rotatory friction welding: a) experimental set-up, b) investigated parameters. a) a) b) Fig. 3. Rotatory friction welding: a) experimental set-up, b) investigated parameters. The parameters were chosen according to the previous work [18] and are designed to prevent unnecessary shortening and to set low temperatures in order to keep the emerging intermetallic phase as small as possible. The steel rotates at a speed of n = 1500 rpm on the spindle side, while on the sledge side the aluminium is rubbed against it with a relative friction path of sR = 4 mm at a friction pressure of pF = 120 MPa. After completion of the friction phase, the upsetting phase takes place with an upsetting pressure of pU = 200 MPa and an upsetting time of tU = 2 s. The resulting welding flash consists of aluminium as only this material is plastically deformed in the process due to its lower strength compared to steel. After the process, the welding flash is removed by machining and the specimens are shortened to a length of 105 mm (42 mm steel, 63 mm aluminium) for the subsequent impact extrusion process. Inductive Heating and Immersion Cooling With the help of an induction coil, the friction-welded hybrid component is heated partially to equalise the significantly different yield stresses of steel and aluminium before the forming process. In order to achieve a favorable flow behavior, an inhomogeneous temperature distribution in the hybrid semi-finished product is necessary. According to simulations and material data, the yield stresses at steel temperatures of 900°C or higher and aluminium temperatures of 20°C are approximately the same level [14]. Due to the time required to heat up and process the component in combination with the thermal conduction during this time, it is not technically possible to reach this ideal equalisation for the forming process. The aim is therefore to achieve the largest possible 594 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 temperature gradient between the two materials. To investigate the influence of the temperature gradient on the material flow, the heating is carried out with and without partial cooling of the aluminium side before forming. During the heating phase, the steel side is in the induction coil, which consists of six rectangular windings. The magnetic field is generated by a 40 kW mid- frequency generator (TRUMPF TruHeat MF 3040) with a frequency range between 5 - 30 kHz. The edge of the aluminium part is at the same level as the lowest coil winding and is cooled by the surrounding water when used with immersion cooling (see Fig. 4). a) b) Fig. 4. Partial heating: a) measured temperatures during induction heating with and without immersion cooling, b) positions of the thermocouples (Tc) and experimental set-up. b) d i i d i h i i h d a) b) Fig. 4. Partial heating: a) measured temperatures during induction heating with and without immersion cooling, b) positions of the thermocouples (Tc) and experimental set-up. The temperature curves shown in Fig. 4 were determined during induction heating without subsequent forming using six thermocouples Type K. Induction heating takes place in segments I to III, where the power is continuously reduced in each stage to counteract overheating close to the surface. Impact Extrusion The forward rod extrusion of the specimens was carried out on a Lasco SPR 500 screw press and took place immediately after the transfer from the induction heating in the tooling system seen in Fig. 5a). To reduce tensile stresses inside the component, a counterpressure pCounter = 160 MPa was generated by four gas springs, which are connected to the counter punch via a transverse beam. The process is designed in such a way that the counterpressure acts on the component shortly before the joining zone passes the extrusion shoulder. To observe the influence of the forming process on the geometric shape of the joining zone, the immersion depth of the punch was varied in a range of 8 mm with a step width of 2 mm. The relative forming path s (see Fig. 5 b)) describes the distance of the joining zone beyond the lower extrusion shoulder. In the case of a negative value for s, the joining zone has not passed the lowest point of the impact extrusion shoulder. b) a) b) Fig. 5. a) Tooling system for forward rod extrusion [19], b) Formation of the joining zone geometry after impact extrusion depending on the relative forming path s. a) b) a) Fig. 5. a) Tooling system for forward rod extrusion [19], b) Formation of the joining zone geometry after impact extrusion depending on the relative forming path s. After forming, the samples were quenched in a water bath. To avoid thermal cracking, first the aluminium was quenched for 1 s, then the steel for 5 s and again the aluminium for 5 s, before the entire specimen was completely immersed for another 20 s. The samples were then cross-sectioned to measure the inner and outer diameter such as the depth of the joining zone to calculate the surface enlargement. The joining zone has a paraboloid geometry, which is why the modified surface area AJZ was calculated approx. using the following equation: 𝐴𝐽𝐽𝜋𝐷𝑖𝐷𝑖𝐻 3𝐷𝑖𝜋𝐷𝑜𝐷𝑖 𝐴𝐴𝐽𝐽𝐽𝐽= 𝜋𝜋⋅( 𝐷𝐷𝑖𝑖 2 ) 6⋅𝐻𝐻2 ൥൬ቀ 𝐷𝐷𝑖𝑖 2 ቁ 2 + 4 ⋅𝐻𝐻൰ 3 2 −ቀ 𝐷𝐷𝑖𝑖 2 ቁ 3 ൩+ 𝜋𝜋 4 (𝐷𝐷𝑜𝑜 2 −𝐷𝐷𝑖𝑖2) (1) (1) At a relative forming path s = -4 mm, the joining zone has not yet fully passed through the impact extrusion shoulder, which is why the paraboloid geometry is not found over the entire cross-section. Inductive Heating and Immersion Cooling After a total of 28 s, the induction heating is finished and the sample is moved out of the coil by a pneumatic cylinder in segment IV so that an automated robot gripper can transfer the sample into the tool with a transfer time of approx. 5 s. By using the immersion cooling, significantly lower temperatures of 84°C instead of 237°C without cooling (Tc6) are set in the aluminium part of the sample, while the steel temperatures at t = 28 s remain the same for both heating strategies reaching nearly 800°C (Tc3). When comparing the material centres (Tc3 and Tc6), a temperature gradient of 694 K can be set with the help of cooling, while only 532 K are achieved without cooling. This corresponds to an increase of approx. 30.5%. An estimated yield stress of about 250 MPa for steel and about 140 MPa (uncooled) and 175 MPa (cooled) for the aluminium is set for strain rate of 𝜑𝜑̇ = 10 1/s and 𝜑𝜑= 0.4. 595 Material Forming - ESAFORM 2023 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Material Forming - ESAFORM 2023 Materials Research Forum LL Materials Research Proceedings 28 (2023) 591-600 https://doi.org/10.21741/9781644902479- Impact Extrusion Therefore, the unformed circular ring (Do - Di) was taken into account in the calculations. Samples with s = +4 were further prepared metallographically. Light microscopic and SEM images as well as EDX analyses were performed using a field emission scanning electron microscope Supra VP 55 to evaluate the cooling effect and the forming sequence on the joining zone geometry and quality as well as the emerging IMC. Results and Discussion The joining zone depth H, which contributes significantly to the surface enlargement, increases with higher relative forming paths s and reaches its maximum for s = +4 (see Fig. 6). In this case 596 Material Forming - ESAFORM 2023 Materials Research Forum LLC Materials Research Proceedings 28 (2023) 591-600 https://doi.org/10.21741/9781644902479-64 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 a joining zone surface of AJZ = 1010.11 mm2 is obtained which corresponds to a surface enlargement of approx. 70% compared to the unaffected, flat joining zone after friction welding or with initially aluminium formed. The measured and calculated values for each variation are shown in Table 2. As the joining zone is passing through the extrusion shoulder, steel forms a funnel, dragging the materially bonded aluminium along with it. The material is drawn more strongly into the centre due to the locally varying forming speed and forms the funnel shape in the absence of subsequent material flow. With increased relative forming path s, the effect mentioned becomes stronger, which is why the shape of the joining zone between steel and aluminium is characterised by a steadily increasing joining zone depth H. Fig. 6. Exemplary images of the joining zone formation for s = [-4, 0, +4]. Fig. 6. Exemplary images of the joining zone formation for s = [-4, 0, +4]. 1. Comparison of the surface enlargement depending on relative forming path s. Table 1. Comparison of the surface enlargement depending on relative forming path s. Initially Formed Material Relative Forming Path s [mm] Inner Diameter Di [mm] Outer Diameter Do [mm] Joining Zone Depth H [mm] Joining Zone Surface AJZ [mm2] Surface Enlargement [%] Aluminium 4 27.5 27.5 0 593.96 0 Steel -4 21.8 27.5 9.5 803.44 35.2 Steel -2 27.5 27.5 12.35 943.67 58.88 Steel 0 27.5 27.5 12.9 968.58 63.07 Steel 2 27.5 27.5 13.3 986.92 66.16 Steel 4 27.5 27.5 13.8 1010.11 70.06 Table 1. Comparison of the surface enlargement depending on relativ The occurring defect in the centre of the specimen is caused by the funnel formation, which usually occurs in monolithic semi-finished products due to a lack of subsequent material flow at the end of the forming process [20]. A locally weak bond in the centre of the joined materials before forming is pre-existent, due to low relative friction speeds in the friction welding process. Combined with an increased forming speed in the centre and differing yield stresses, a detachment of the two materials is very likely. 596 The defect can mostly be avoided if the extrusion takes place with high friction between the extrusion die and the specimen [21]. In this regard the use of a counterpressure can set the required stress state [11-13]. Since the gap was nevertheless observed during the tests carried out, it can be assumed that the set of a counterpressure pCounter = 160 MPa is not sufficient to completely compensate the tensile stresses acting in the joining zone. The greater temperature gradient caused by the immersion cooling (see Fig. 4) provides a better equalisation of the yield stresses, thus a lower deformation resistance ratio and therefore a less pronounced paraboloid shape. Furthermore, the constriction of the aluminium is largely prevented (see Fig. 7) so that, unlike the sample without cooling, no gap extends over the entire circumference of the component surface near the joining zone. Due to the improved material flow, the central defect is also slightly reduced from 132 µm to 53 µm, but cannot be completely prevented. The influence of the partial cooling is also reflected in the measured hardness (HV0.1), 597 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 which show an average increase of 12 HV from 61 HV without cooling to 73 HV with cooling for the aluminium. The hardnesses in the steel are subject to relatively large deviations, but also increased slightly on average by 8 HV from 283 HV to 291 HV. which show an average increase of 12 HV from 61 HV without cooling to 73 HV with cooling for the aluminium. The hardnesses in the steel are subject to relatively large deviations, but also increased slightly on average by 8 HV from 283 HV to 291 HV. g y g y Fig. 7. Metallographic and SEM images of the joining zone (s = +4 mm) without and with cooling with a counter pressure of pCounter = 160 MPa. Fig. 7. Metallographic and SEM images of the joining zone (s = +4 mm) without and with cooling with a counter pressure of pCounter = 160 MPa. Regardless of the used heating strategy, the formed IMCs have been influenced by the surface enlargement leading to a relatively homogenous structure. The thickness in the edge area as well as in the descending area is varying between 0.6 to 0.8µm. Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Acknowledgments The results presented in this paper were obtained within sub-project B03 of the Collaborative Research Centre 1153 “Process chain to produce hybrid high performance components with Tailored Forming” funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - SFB 1153 TP B03 - 252662854. The authors thank the DFG for the financial support of this project and also the Institute of Materials Science for the SEM/EDX investigations. Summary y In this study, a forward rod extrusion process was used which, based on preliminary work, focused on the forming sequence and the use of partial cooling for the aluminium side. The variation of the forming path showed a material flow-related increase of the surface enlargement of up to 70%. Furthermore, the complete passing of the joining zone through the impact extrusion shoulder resulted in defects near the surface as well as on the interior, which could be reduced with the help of the improved material flow through immersion cooling. A homogenous formation of the IMC is ensured in the enlarged areas. Partial detachments or cracking of the IMC was observed regardless of cooling in the areas with increased surface area, especially in the centre at the highest material flow rates. For future work, the counter pressure should be increased further in order to compensate the tensile stresses that occur for a defect-free bond. Furthermore, mechanical tests should be carried out to prove that the enlarged surface also improves the bond strength of the joining zone. To investigate the possible adjustable maximum temperature gradients, selecting other cooling strategies such as air-spray cooling should be considered. 596 Near the central area, the brittle IMCs are destructed after forming due to the detachment between the two materials. The IMC thicknesses in the edge area are approx. in the same size range compared to the thicknesses in the central area, which is why no thinning of the IMC can be certainly observed due to the surface enlargement. However, since the IMC does not exceed the critical thickness of 1 µm [16] combined with the increase in surface area compared to a flat joining zone, a potential improvement of the bond strength can be assumed. As expected, the chemical composition in the joining zone was not influenced by the cooling. An EDX line scan of the specimen without cooling at a forming path of s = +4 mm (see Fig. 8) is exemplary shown in Fig. 8. A diffusion and hence a deposition of manganese and silicon into the area of the joining zone with a concentration of up to 5 wt.% is seen, indicating the existing IMC. j g p g g Fig. 8. EDX analysis of the descending area for a specimen without cooling (s = +4 mm). Fig. 8. EDX analysis of the descending area for a specimen without cooling (s = +4 mm). 598 Material Forming - ESAFORM 2023 Materials Research Forum LLC Materials Research Proceedings 28 (2023) 591-600 https://doi.org/10.21741/9781644902479-64 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Proceedings 28 (2023) 591-600 Material Forming - ESAFORM 2023 Materials Research Forum LLC https://doi.org/10.21741/9781644902479-64 Materials Research Forum LLC org/10 21741/9781644902479 64 https://doi.org/10.21741/9781644902479-64 [10] B.-A. Behrens, J. Uhe, I. Ross, J. Ursinus, T. Matthias, S. Bährisch, Tailored Forming of hybrid bulk metal components, Int. J. Mater. Form. 15 (2022) 42. https://doi.org/10.1007/s12289- 022-01681-9 [11] D.-C. Ko, B.-M. Kim, The prediction of central burst defects in extrusion and wire drawing, J. Mater. Process. Technol. 102 (2000) 19-24. https://doi.org/10.1016/S0924-0136(99)00461-6 [12] H. Hoche, A. Balser, M. Oechsner, A. Franceschi, P. Groche, Enhancement of the residual stresses of cold full-forward extruded parts by application of an active counter punch, Materialwissenschaft und Werkstofftechnik 50 (2019) 669-681. https://doi.org/10.1002/mawe.201900050 p g [13] C. Soyarslan, A.E. Tekkaya, Prevention of Internal Cracks in Forward Extrusion by Means of Counter Pressure: A Numerical Treatise, Steel Res. Int. 80 (2009) 671-679. https://doi.org/10.2374/SRI08SP170 p g [14] C. Büdenbender, I. Ross, H. Wester, A. Zaitsev, B.-A. Behrens, Numerical Investigation of an Extruded Shaft for High Temperature Applications Manufactured by Tailored Forming, in: Production at the leading edge of technology. Lecture Notes in Production Engineering, Springer, Berlin, Heidelberg, 2021, pp. 182-192. https://doi.org/10.1007/978-3-662-62138-7_19 [15] P. Gumm, Kombination von Umformung und Kaltpreßschweißen beim Fließpressen und Rohrziehen (Dissertation), VDI Verlag GmbH, Technische Hochschule Braunschweig, 1964. ( ) g g [16] S. Herbst, H.J. Maier, F. Nürnberger, Strategies for the Heat Treatment of Steel-Aluminium Hybrid Components, HTM J. Heat Treat. Mater. 73 (2018) 268-282. https://doi.org/10.3139/105.110368 g g Hybrid Components, HTM J. Heat Treat. Mater. 73 (2018) 268-282. https://doi.org/10.3139/105.110368 [17] A. Piwek, J. Uhe, J. Peddinghaus, I. Ross, B.-A. Behrens, Comparison of the joining zone development of hybrid semi-finished products after different extrusion processes, 31st International Conference on Metallurgy and Materials, Orea Congress Hotel Brno, Czech Republic, EU, May 18 - 19, 2022. https://doi.org/10.37904/metal.2022.4398 p y p g [18] B.-A. Behrens, D. Duran, T. Matthias, I. Ross: Enhancement of the interface friction welded steel-aluminium joints, Prod. Eng. Res. Devel. (2021) 169-176. https://doi.org/10.1007/s11740- 020-00994-5 [19] B.-A. Behrens, R. Goldstein D. Duran, Role of Thermal Processing in Tailored Forming Technology for Manufacturing Multi-Material Components, Heat Treat 2017: Proceedings of the 29th ASM Heat Treating Society Conference, October 24-26, Columbus, Ohio, USA, 2017 [20] H.S. Valberg, M. Lefstad, A.L. de Moraes Costa, On the Mechanism of Formation of Back- End Defects in the Extrusion Process, Procedia Manuf. 47 (2020) 245-252. https://doi.org/10.1016/j.promfg.2020.04.207 [21] I. Balasundar, T. 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https://openalex.org/W2154060112	https://www.frontiersin.org/articles/10.3389/fmicb.2010.00130/pdf	English	null	Circadian Clock Proteins in Prokaryotes: Hidden Rhythms?	Frontiers in microbiology	2,010	cc-by	9,764	Introduction Apart from the circadian clock rhythm described and exten sively studied in S. elongatus (Ishiura et al., 1998) there is only little evidence of circadian rhythms in other cyanobacterial spe cies (Aoki and Onai, 2009) and even less in other prokaryotes that contain kai genes. The only other bacterium where the presence of a circadian-like timing mechanism was described is the purple bacterium Rhodobacter sphaeroides (Min et al., 2005). However, other prokaryotes apart from cyanobacteria and purple bacte ria might be influenced by the daily Earth’s life cycle leading to enhanced fitness by improving their ability to adapt to extrinsic influences, specifically the daily changes in environmental factors such as light, temperature, and humidity (Woelfle et al., 2004). This might explain, why several other prokaryotes acquired the kai genes and components of the circadian system in their genomes. Insight into the roles of the circadian clock proteins in other prokaryotes would also provide a better understanding of their evolutionary his tory. These Kai proteins might be involved in metabolic processes, chromosome compaction (Woelfle et al., 2007), cell division (Mori et al., 1996; Mori, 2009), and UV stress resistance (Garcia-Pichel, 1998; Dvornyk and Nevo, 2003) as described for cyanobacteria or may have evolved divergent functions. Many living organisms from bacteria to eukaryotes have evolved internal mechanisms to synchronize their physiological functions with the Earth’s rotation cycle. These internal timing mechanisms of about 24 h are named circadian cycles. In the molecular context, circadian cycles are self-sustained biochemical oscillations that are temperature compensated and persist in absence of external envi ronmental cues. This endogenous mechanism is able to respond to changes in the environment (like intensities of light and dark) and to entrain its phase to the local environmental cues. The distribu tion of circadian clocks in eukaryotic organisms is wide (Jolma et al., 2010). In contrast in prokaryotes the circadian clock genes are found only in some groups of bacteria (e.g., Cyanobacteria, Proteobacteria, Bacteroidetes) and archea (see Table 1 and Dvornyk et al., 2003). Among prokaryotes the circadian mechanism was first reported in the cyanobacterium Synechococcus elongatus PCC 7942 (Ishiura et al., 1998), which since then became a model to study this complex system. The circadian clock core genes in cyanobacteria are named kaiABC (kai after the Japanese word kaiten for a cycle or “turning of the heavens”). Maria Loza-Correa1,2, Laura Gomez-Valero1,2 and Carmen Buchrieser1,2* Maria Loza-Correa1,2, Laura Gomez-Valero1,2 and Carmen Buchrieser1,2* 1 Institut Pasteur, Unité de Biologie des Bactéries Intracellulaires, Paris, France 2 CNRS URA 2171, Paris, France 1 Institut Pasteur, Unité de Biologie des Bactéries Intracellulaires, Paris, France 2 CNRS URA 2171, Paris, France Circadian clock genes are vital features of eukaryotes that have evolved such that organisms can adapt to our planet’s rotation in order to anticipate the coming day or night as well as unfavorable seasons. This circadian clock uses oscillation as a timekeeping element. However, circadian clock mechanisms exist also in prokaryotes. The circadian clock of Cyanobacteria is well studied. It is regulated by a cluster of three genes: kaiA, kaiB, and kaiC. In this review, we will discuss the circadian system in cyanobacteria, and provide an overview and updated phylogenetic analysis of prokaryotic organisms that contain the main circadian genes. It is evident that the evolution of the kai genes has been influenced by lateral transfers but further and deeper studies are needed to get an in depth understanding of the exact evolutionary history of these genes. Interestingly, Legionella pneumophila an environmental bacterium and opportunistic human pathogen that parasitizes protozoa in fresh water environments also contains kaiB and kaiC, but their functions are not known. All of the residues described for the biochemical functions of the main pacemaker KaiC in Synechococcus elongatus are also conserved in the L. pneumophila KaiC protein. Correspondence: Correspondence: Carmen Buchrieser, Biology of Intracellular Bacteria, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France. Carmen Buchrieser, Biology of Intracellular Bacteria, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France. e-mail: carmen.buchrieser@pasteur.fr Keywords: circadian clock genes, Legionella, archea, proteobacteria, cyanobacteria, evolution Review Article published: 06 December 2010 doi: 10.3389/fmicb.2010.00130 Edited by: Elizabeth L. Hartland, University of Melbourne, Australia Reviewed by: Elizabeth L. Hartland, University of Melbourne, Australia Antje Flieger, Robert Koch Institute, Germany www.frontiersin.org Review Article published: 06 December 2010 doi: 10.3389/fmicb.2010.00130 Introduction Cyanobacteria play an important role in the history of the Earth as the primary producers of oxygen (Tomitani et al., 2006). Since their appearance on Earth, they have evolved different strategies to survive the harsh environmental conditions they have to face like UV radiation, changes in temperature or changes in the redox state of the atmosphere (Garcia-Pichel, 1998). Furthermore, cyanobacteria are organisms that depend on the light/dark cycle, allowing oxygenic pho tosynthesis to occur during the day, in the presence of sunlight, and oxygen-sensitive nitrogen fixation during the dark period. Circadian clock systems have evolved as important tools that have been pivotal for their adaptation to a wide range of environmental conditions where cellular functions are linked to photochemical processes. Surprisingly, L. pneumophila, an environmental bacterium para sitizing aquatic protozoa, but also an important human pathogen causing a severe pneumonia called Legionnaires’ disease, contains in its genome genes encoding homologues of KaiB and KaiC (Cazalet et al., 2004; Albert-Weissenberger et al., 2007). These genes are conserved in the sequenced L. pneumophila genomes, but KaiC of L. pneumophila strain Lens contains a frameshift at its N-terminus, that truncates the first walker motif. In contrast, these genes do not belong to the core genome of the genus Legionella, as Legionella longbeachae, does not encode putative circadian clock proteins (Cazalet et al., 2010) neither Legionella micdadei (unpublished) December 2010 \| Volume 1 \| Article 130 \| 1 www.frontiersin.org Circadian clock genes in prokaryotes Loza-Correa et al. Table 1 \| Distribution of KaiA, KaiB, and KaiC homologues in completely sequenced prokaryotic genomes. Family Species with best KaiC-BLASTp hit kaiA kaiB kaiC Genera incertae sedis Candidatus Methanoregula boonei − + + Archaeoglobaceae Archaeoglobus profundus − − + Methanocaldococcaceae Methanocaldococcus jannaschii − − + Methanopyraceae Methanopyrus kandleri − − + Halobacteriaceae Halalkalicoccus jeotgali − − + Thermofilaceae Thermofilum pendens − − + Desulfurococcaceae Thermosphaera aggregans − − + Thermococcaceae Pyrococcus horikoshii − − + Synechococcaceae Synechococcus elongatus + + + Oscillatoriaceae Oscillatoria sp. + + + Nostocaceae Nodularia spumigena + + + Unclassified Cyanobacteria Acaryochloris marina + + + Prochlorococcaceae Prochlorococcus marinus − + + Bradyrhizobiaceae Bradyrhizobium sp. In red Archea, in blue Proteobacteria, in green Cyanobacteria, in orange Bacteroidetes, and in gray other bacteria. (+) Indicates presences of the gene sequence; (−) indicates absence of the gene sequence in the genome. Introduction BTAi1 − + + Rhodospirillaceae Rhodospirillum rubrum − + + Phyllobacteriaceae Mesorhizobium opportunistum − + + Rhodobacteraceae Rhodobacter sphaeroides − + + Chromatiaceae Allochromatium vinosum − + + Ectothiorhodospiraceae Halorhodospira halophila − + + Legionellaceae Legionella pneumophila − + + Methylobacteriaceae Methylobacterium populi − + + Geobacteraceae Geobacter sp. − + + Nannocystineae Haliangium ochraceum − + + Desulfohalobiaceae Desulfonatronospira thiodismutans − + + Xanthomonadaceae Stenotrophomonas maltophilia − − + Myxococcaceae Anaeromyxobacter dehalogenans − − + Aurantimonadaceae Aurantimonas manganoxydans − − + Rhizobiaceae Agrobacterium tumefaciens − − + Caulobacteraceae Caulobacter segnis − − + Sphingomonadaceae Sphingobium japonicum − − + Rhizobiaceae Sinorhizobium medicae − − + Beijerinckiaceae Methylocella silvestris − − + Hyphomicrobiaceae Rhodomicrobium vannielii − − + Burkholderiales Burkholderia sp. − − + Comamonadaceae Variovorax paradoxus − − + Oxalobacteraceae Herbaspirillum seropedicae − − + Unclassified Epsilonproteobacteria Nitratiruptor sp. − − + Halomonadaceae Chromohalobacter salexigens − − + Pseudomonadaceae Pseudomonas putida − − + Desulfomicrobiaceae Desulfomicrobium baculatum − − + Myxococcaceae Myxococcus xanthus − − + Cystobacterineae Stigmatella aurantiaca − − + Chlorobiaceae Chloroherpeton thalassium − + + Cytophagaceae Spirosoma linguale − + + Chitinophagaceae Chitinophaga pinensis − + + Flavobacteriaceae Flavobacterium johnsoniae − + + Chloroflexaceae Roseiflexus castenholzii − + + Verrucomicrobia Chthoniobacter flavus − + + Planctomycetaceae Rhodopirellula baltica − − + Thermotogaceae Thermotoga petrophila − − + \| Distribution of KaiA, KaiB, and KaiC homologues in completely sequenced prokaryotic genomes. In red Archea, in blue Proteobacteria, in green Cyanobacteria, in orange Bacteroidetes, and in gray other bacteria. (+) Indicates presences of the gene sequence; (−) indicates absence of the gene sequence in the genome. December 2010 \| Volume 1 \| Article 130 \| 2 Frontiers in Microbiology \| Cellular and Infection Microbiology Circadian clock genes in prokaryotes Loza-Correa et al. reported for the purple bacterium Rhodobacter sphaeroides (Min et al., 2005). This proteobacterium displays circadian oscillations under aerobic conditions. Rh. Sphaeroides contains the kaiB and kaiC cyanobacterial homologous genes, but lacks the kaiA gene. One possible explanation of the absence of KaiA in Rh. sphaeroides could be that KaiB and KaiC have evolved functions different from those in cyanobacteria, and thus KaiA is not necessary anymore (Min et al., 2005). So far, the work of Min et al. (2005) has been the first report with evidence of a circadian rhythm in a proteobacte rium. Circadian systems in Cyanobacteria The core element of the circadian system in the model organism S. elongatus PCC 7942 (hereafter S. elongatus) is a cluster of three tandemly located genes called: kaiA, kaiB, and kaiC that are essential for circadian rhythm in Synechococcus. Among these genes, kaiB and kaiC operate with the same promoter (Ishiura et al., 1998) and both genes are conserved among many other prokaryotes including sev eral archea (Dvornyk et al., 2003). In some cyanobacterial genomes there are also paralogs of kaiB and kaiC at other loci (Dvornyk et al., 2003; Aoki and Onai, 2009). For example, Synechocystis sp. strain PCC6803 contains one copy of kaiA but kaiB and kaiC are present in triplicates in its genome, located in four different loci: kaiABC, kaiC2B2, kaiB3, and kaiC3 (Aoki and Onai, 2009). In con trast, kaiA is missing in many prokaryotic genomes that contain kaiBC. However, it has been shown that the marine cyanobacterium Prochlorococcus displays circadian cycles, despite the fact that it con tains only the kaiB and kaiC genes but not kaiA, which has suffered a stepwise deletion, together with significant genome streamlining (Holtzendorff et al., 2008). Natural Prochlorococcus populations and laboratory cultures are strongly synchronized by the alternation of day and night, displaying 24-h rhythms in DNA replication. It was shown that the KaiB and KaiC proteins fulfill their known bio chemical functions and that a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus may be sufficient for biological timing suited to the very stable environment where Prochlorococcus is found (Axmann et al., 2009). This finding opens the possibility that other prokaryotes like proteobacteria or archea that contain the kaiBC genes could also display an oscillator machinery similar to the one in Prochlorococcus. It was believed that the Kai proteins were ubiquitous among cyanobacteria, but an “exception to the rule” was reported when the genome of Gloeobacter violaceus PPC7421 was sequenced (Nakamura et al., 2003). This cyanobacterium lacks the kaiABC genes as well as the sasA, cikA, and pex genes, which are other major components of the clock system (see below). So far it is not known if G. violaceus displays circadian rhythmicity, possibly through another, not yet identi fied system, or not. However, this organism also shows ancestral characteristics for oxygenic photosynthesis and lacks thylakoids (Nakamura et al., 2003) characteristics reflecting its phylogenetic distance to other cyanobacteria. Circadian systems in Cyanobacteria Elucidation of the circadian clock behavior in purple bacteria as well as efforts to understand a possible circadian system in other non-photosynthetic proteobacteria may also help to get a better understanding on its evolution and origin(s). In a first approach to better understand the distribution and evolution of the circadian clock core genes, we have undertaken a search for the presence of kaiC genes in all completely sequenced prokaryotic and archeal genomes available in the NCBI database (http://www.ncbi.nlm. nih.gov/sutils/genom_table.cgi). To search for KaiC proteins we used the S. elongatus KaiC amino acid sequence as seed sequence and Blastp similarity searches. All hits covering at least 80% of the length of the S. elongatus KaiC protein sequence with a minimum e-value of e−5 were taken into account. Finally, the sequence show ing the best hit with S. elongatus KaiC of each family of bacteria or archea, containing KaiC was chosen as representative for further analysis. In total 53 sequences belonging each to different species were retained (Table 1). To date (September 2010) the number of complete microbial genome sequences available in the NCBI database has increased to 1408 (1332 bacteria and 76 archea) allowing to identify additional organisms containing kai genes in their genome. Dvornyk et al. (2003) reported the presence of kai genes in four groups of organisms: Cyanobacteria, Proteobacteria, Archea, and Chloroflexi. The list of organisms has now increased in each of these groups (Table 1) and in addition we identified kai genes in the phylum Bacteroidetes. Furthermore, we have found independent cases of occurrence of the kai genes in different families of organisms that we named “other bacteria,” which com prise Chlamydiae, Planctomycetes, and Acidobacteria (Table 1). Interestingly, only Cyanobacteria (except Prochlorococcus sp.) encode KaiA in their genomes, but none of the other prokaryo tes sequenced to date contain KaiA, even when they encode KaiB and KaiC or KaiC alone. KaiB sequences are present in all cyanobacteria sequenced and Bacteroidetes, but only in some proteobacteria. KaiB is present in some taxa belonging to the α-proteobacteria, γ-proteobacteria, and δ-proteobacteria, but not in β- or ε- proteobacteria listed in Table 1. This irregularity in the occurrence of circadian clock proteins in proteobacteria has also been described by Dvornyk et al. (2003). Here we report an interesting, new example, the family Legionellaceae. As stated above only L. Introduction Rhythmic oscillations have also been reported in an other purple bacteria, Rhodospirillum rubrum (Van Praag et al., 2000), however it remains unknown if the gene expression in this organism is rhythmically controlled or not and whether the cyanobacterial homologs of kaiB and kaiC are involved. nor Legionella drancourtii. Intrigued by the identification of these proteins in L. pneumophila, we investigated the circadian clock genes in prokaryotes further. Here we provide an updated overview of prokaryotic organisms whose genome contains kai genes and analyze their evolutionary history. We then discuss what is known about the biochemistry of the Kai proteins and their roles reported for cyanobacteria and conclude with hypothesis of the role they could play in L. pneumophila. Circadian systems in Cyanobacteria pneumophila strains contain both, genes encoding homologues of KaiC and KaiB but none of the other Legionella species of which the genome sequence is known. Thus, L. pneu mophila is the first bacterial human pathogen reported to contain www.frontiersin.org Input pathway and its components The input pathway of the circadian clock transmits signals from the environment to entrain the oscillator. The signals that can be sensed are usually light and temperature. For example, the circa dian clock of S. elongatus can be reset by signals such as light and dark pulses (Schmitz et al., 2000) or pulses of high kaiC expression levels (Ishiura et al., 1998). The main components of the input pathway in S. elongatus are CikA (circadian input kinase) that sense the cellular redox state and environmental light intensity Figure 1 \| Schema of the molecular mechanisms of the circadian clock in S. elongatus. The input pathway responds to the environmental signals like light and temperature to entrain the circadian oscillator. Both CikA and LdpA can sense changes in the redox state of the cell. CikA interacts with quinones through its pseudo receiver domain. The components of the input pathway influence the phosphorylation state of the KaiC hexameric complex. The KaiA homodimer complex binds to KaiC and stimulates its phosphorylation. KaiC is first autophosphorylated at the T432 conserved residue and reaches the highest phosphorylation state when on each KaiC monomer the T321 and S431 residues are phosphorylated. Then KaiB tetramers bind to the KaiC complex and also SasA binds to the complex to transmit the signals downstream and to shift the global gene expression, regulate metabolic functions and regulate chromosomal compaction. The circadian cycle will have transcriptional–translational feedback from the global gene expression increasing its robustness. Finally the dephosphorylation state starts and in the early phase KaiC monomer shuffling occurs that might help to synchronize the clock with the environmental cues. The KaiC hexameric complex first loses the phosphorylation on the T432 and then in the S431 before the cycle starts again. Figure 1 \| Schema of the molecular mechanisms of the circadian clock in S. elongatus. The input pathway responds to the environmental signals like light and temperature to entrain the circadian oscillator. Both CikA and LdpA can sense changes in the redox state of the cell. CikA interacts with quinones through its pseudo receiver domain. The components of the input pathway influence the phosphorylation state of the KaiC hexameric complex. The KaiA homodimer complex binds to KaiC and stimulates its phosphorylation. KaiC is first autophosphorylated at the T432 conserved residue and reaches the highest phosphorylation state when on each KaiC monomer the T321 and S431 residues are phosphorylated. Circadian systems in proteobacteria Although cyanobacteria have become a major model system for analyzing clock phenomena in prokaryotes, efforts have been undertaken to get insight into the possible circadian rhythms of other prokaryotes. Indeed, recently a circadian oscillator has been December 2010 \| Volume 1 \| Article 130 \| 3 www.frontiersin.org Circadian clock genes in prokaryotes Loza-Correa et al. downstream biochemical processes. In the output pathway, the key players in S. elongatus are SasA (Iwasaki et al., 2000), RpaA (Takai et al., 2006), and LabA (Taniguchi et al., 2007) (Figure 1). the circadian genes (kaiBC). It will be an interesting question to answer whether they are involved in virulence and intracellular survival and replication of L. pneumophila or if they enhance its fitness in the environment. In bacteria, the Kai proteins constitute the core for the circadian system, but they do not have homologues in the genomes of mam mals, insects or fungi. However, both prokaryotic and eukaryotic circadian systems show autoregulatory features that involve a nega tive feedback loop in which the clock genes encode proteins that repress their own transcription (Ishiura et al., 1998; Glossop et al., 1999; Cheng et al., 2001). Understanding the biochemistry of the circadian clock components: the example S. elongatus Many different groups have been investigating the biological clock machinery of the cyanobacterial model S. elongatus PCC 7942 to get major insight into the mechanisms and the role of this system. In general, this circadian system shares three major components: (1) a central oscillator generating the fundamental rhythm of approxi mately 24 h; in S. elongatus the oscillator core consists of the KaiA, KaiB, and KaiC proteins (Ishiura et al., 1998); (2) an input pathway conveying environmental signals to the oscillator and modifying the oscillation to synchronize it precisely with the daily cycle. In S. elongatus the proteins CikA, LdpA, and Pex are key components for this input pathway (Kutsuna et al., 1998; Schmitz et al., 2000; Katayama et al., 2003; Ivleva et al., 2006); (3) an output pathway that relays temporal information from the oscillator to a variety of Input pathway and its components Then KaiB tetramers bind to the KaiC complex and also SasA binds to the complex to transmit the signals downstream and to shift the global gene expression, regulate metabolic functions and regulate chromosomal compaction. The circadian cycle will have transcriptional–translational feedback from the global gene expression increasing its robustness. Finally the dephosphorylation state starts and in the early phase KaiC monomer shuffling occurs that might help to synchronize the clock with the environmental cues. The KaiC hexameric complex first loses the phosphorylation on the T432 and then in the S431 before the cycle starts again. Figure 1 \| Schema of the molecular mechanisms of the circadian clock in chema of the molecular mechanisms of the circadian clock in Figure 1 \| Schema of the molecular mechanisms of the circadian clock in S. elongatus. The input pathway responds to the environmental signals like light and temperature to entrain the circadian oscillator. Both CikA and LdpA can sense changes in the redox state of the cell. CikA interacts with quinones through its pseudo receiver domain. The components of the input pathway influence the phosphorylation state of the KaiC hexameric complex. The KaiA homodimer complex binds to KaiC and stimulates its phosphorylation. KaiC is first autophosphorylated at the T432 conserved residue and reaches the highest phosphorylation state when on each KaiC monomer the T321 and S431 residues December 2010 \| Volume 1 \| Article 130 \| 4 Frontiers in Microbiology \| Cellular and Infection Microbiology Loza-Correa et al. Circadian clock genes in prokaryotes KaiA forms a homodimer that interacts directly with hexamers of KaiC by binding at the C-terminal tail (Kim et al., 2008). This interaction stimulates its phosphorylation and inhibits dephospho rylation steps whereas tetramers of KaiB antagonize the effects of KaiA (Xu et al., 2003). In the in vitro system, Kai protein complexes assemble and disassemble dynamically over the KaiC phosphoryla tion cycle (Kageyama et al., 2006; Mori et al., 2007). In the initial step hexamers of KaiC are in unphosphorylated form. It associates with KaiA and subsequently KaiC autophosphorylates at residue T432 and then the double phosphorylated forms of KaiC appear (T432-S431-KaiC). This is the highest level of phosphorylation, KaiB binds to KaiC and when the KaiABC complex predominates the dephosphorylation phase starts. Input pathway and its components In the early phase of dephos phorylation “monomer shuffling” occurs among the KaiC hexam ers probably helping to synchronize KaiC with the environmental cues (Ito et al., 2007). Finally, KaiC appears monophosphorylated at residue S431, dissociates from KaiB and the cycle starts a new (Nishiwaki et al., 2004, 2007; Rust et al., 2007) (Figure 1). Recently a third phosphorylation site, a threonine residue at position 42, has been reported to also be important to maintain rhythmicity (Xu et al., 2009). The phosphorylation state of the oscillator has been proposed as the pacemaker of the circadian clock, but there is evidence that circadian gene expression persists also when the phosphorylation cycle is disrupted (Kitayama et al., 2008). (Ivleva et al., 2006) and LdpA (light-dependent period) that is also sensitive to the redox state of the cell. CikA senses light and dark from the environment through the direct interaction with quinone molecules whose redox state depends on light and the metabolic redox state of the bacteria (Ivleva et al., 2006). CikA has a GAF domain, histidine protein kinase (HPK) and a pseudo receiver domain (PsR) necessary for quinone sensitivity (Mutsuda et al., 2003; Ivleva et al., 2006). (Ivleva et al., 2006) and LdpA (light-dependent period) that is also sensitive to the redox state of the cell. CikA senses light and dark from the environment through the direct interaction with quinone molecules whose redox state depends on light and the metabolic redox state of the bacteria (Ivleva et al., 2006). CikA has a GAF domain, histidine protein kinase (HPK) and a pseudo receiver domain (PsR) necessary for quinone sensitivity (Mutsuda et al., 2003; Ivleva et al., 2006). Co-purification assays showed that in addition to the Kai pro teins CikA and LdpA form part of the periodosome (the heterom ultimeric complex necessary to sustain circadian rhythms; Golden, 2004) together with a protein of the output pathway named SasA (Ivleva et al., 2005). LdpA entrains the clock with the intensity of the light through sensing the changes in electron transport and molecules like O2 or reactive oxygen species (Ivleva et al., 2005). The LdpA protein contains an HcyB domain and two specific, conserved terminal domains and belongs to the ferredoxin family of proteins (Katayama et al., 2003). cikA mutants are defective in the ability to reset the phase of the rhythm after a dark pulse and affect phosphorylation of KaiC (Ivleva et al., 2006). Output pathway and its components Phosphorylation phase and ATPase activity levels of the oscil lator complex determine the information transmitted to the downstream transcriptional regulatory system (Nishiwaki et al., 2004; Dong et al., 2010). This regulatory system is constituted of the SasA (Synechococcus adaptive sensor A)-RpaA (regulator of phycobilisome-associated) two-component system (Takai et al., 2006). The HPK SasA forms a complex with KaiC by binding KaiC through its KaiB-like sensory domain (Iwasaki et al., 2000) leading to autophosphorylation and the transfer of a phosphate to the DNA binding response regulator RpaA (Takai et al., 2006). Both, SasA autophosphorylation and the SasA-to-RpaA phospho transfer are modulated according to the KaiC phosphorylation state. Consequently the target genes of RpaA can be activated or repressed in a circadian manner. Thus the KaiC-SasA-RpaA inter action is so far the major positive pathway known to regulate the circadian shift in gene expression (Takai et al., 2006). Mutants in sasA grow slowly in comparison to the wild type bacteria in a light/dark cycle (Iwasaki et al., 2000) and produce unstable short periods of transcription rhythms (Takai et al., 2006). Null mutants of rpaA show arrhythmia under continues light conditions (Takai et al., 2006). Input pathway and its components In contrast ldpA mutants show a conditional alteration in the circadian period as compared to the wild type due to an insensitivity to light gradi ent that normally modulates the circadian period resulting in a lengthening of the period at low light intensities. Thus the LdpA protein seems to modulate the circadian clock as an indirect func tion of light intensity by sensing changes in cellular physiology (Katayama et al., 2003). Another protein involved in the input pathway is Pex (period-extender) a transcriptional repressor of KaiA (Kutsuna et al., 1998). Constitutive expression of pex leads to a prolongation of the circadian period to 28 h. In contrast cells lacking pex, show a 1-h shorter circadian period due to the increase of KaiA that leads to a faster phosphorylation of KaiC (Kutsuna et al., 1998). www.frontiersin.org The Kai core proteins KaiC is the central oscillatory protein. Its primary sequence con tains a double KaiC domain (CI and CII) and each domain con tains a Walker motif that can bind ATP (Nishiwaki et al., 2000). KaiC belongs to the RecA/DnaB protein superfamily. Although the members of this family have the ability to bind DNA, KaiC lacks a DNA binding motif and does not show affinity for single or double stranded DNA. However, KaiC can bind forked DNA (Mori et al., 2002). KaiC is the only of the three proteins with enzymatic activity. It was reported to function as kinase, auto phosphorylase, and ATPase (Nishiwaki et al., 2007; Terauchi et al., 2007) and that its activity is modulated through the interaction with KaiA and KaiB (Iwasaki et al., 2002; Kitayama et al., 2003). Indeed, self-sustainable oscillation of KaiC phosphorylation could be reconstituted in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate. The period of the in vitro oscillation was stable despite temperature change, and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro (Nakajima et al., 2005). The KaiC phosphor ylation state persists in absence of transcriptional feedback and protein synthesis (Tomita et al., 2005), however the transcription and translation feedback increases the robustness of the circadian clock system (see Johnson et al., 2008). An alternative output pathway by which the circadian clock modulates circadian gene expression is LabA (low-amplitude and bright). LabA is needed for negative feedback regulation of KaiC (Taniguchi et al., 2007). Mutants in labA increase the levels of circa dian gene expression due to the high levels of non-regulated-KaiC resulting in a low amplitude phenotype. In contrast, labA overex pression results in low circadian gene expression (Taniguchi et al., 2007). Furthermore, it has been suggested that the LabA pathway is implicated in chromosomal compaction (Woelfle et al., 2007). Both pathways LabA and SasA work in parallel to regulate the expression December 2010 \| Volume 1 \| Article 130 \| 5 www.frontiersin.org Circadian clock genes in prokaryotes Loza-Correa et al. restricted to the dark period. In addition, DNA replication also remains constant during the circadian cycle suggesting that the most evident target is cytokinesis (Mori et al., 1996; Mori, 2009; Dong et al., 2010). The Kai core proteins Much knowledge to better understand the biological significance of the circadian clock gating different cell functions has been gained in the last years, but further studies are needed to complete our understanding of the impact of the prokaryotic circadian clock on the global physiology of the cell and research should be expanded on putative circadian clocks of other prokaryotes. of kaiBC in S. elongates. RpaA is situated downstream in this cascade to generate the robust modulation of circadian gene expression (Kondo, 2007) (Figure 1). Adaptations to changes in the intensities of lighti One of the first physiological evidence that suggested the pres ence of circadian clock mechanisms in cyanobacteria was the observation that nitrogen fixation onset occurs in a rhythmic way (Grobbelaar et al., 1986). Apart from photosynthesis and nitro gen fixation, circadian clocks regulate amino acid uptake (Chen et al., 1991), cell division (Sweeney and Borgese, 1989; Mori et al., 1996), global patterns of gene expression (Liu et al., 1995), and topological chromosomal compaction (Smith and Williams, 2006). Circadian expression in S. elongatus was shown to be pervasive using bioluminescence to monitor the promoter activity of many different genes. Liu et al. (1995) reported that no special set of genes controlled by the circadian clock exists, but that many of these genes probably are regulated by cis- and trans-acting regula tory factors that can incorporate non-specific circadian control and circadian regulation from components in the downstream circadian output pathway. Many mechanisms that impact the functioning of the cell and how they make the circadian clock work, remain to be discovered and deciphered, but new parts in the puzzle are added constantly. For example it was known for many years that the circadian clock gates the cell cycle process but the mechanisms that allow this phe nomenon were unknown (Mori et al., 1996; Mori and Johnson, 2000). However, very recently Dong et al. (2010) reported that the circadian clock has a checkpoint on cell division that can inhibit cytokinesis at specific points of the circadian cycle. Normally, the “gate” is open so cell division can happen independently of the circadian clock (Mori et al., 1996; Mori, 2009), but the gate closes when high levels of KaiC ATPase activity are present. Then, the signal downstream is transmitted through the SasA-RpaA pathway and it results in the inhibition of the midcell FtsZ ring assembly, blocking cell division until the levels of ATPase activ ity decrease (Dong et al., 2010). The biological implication of the circadian checkpoint in cell division may impact in different ways by protecting other cellular functions that can be damaged as result of cell division in vulnerable conditions (Dong et al., 2010). It is important to notice that in S. elongatus the gate occurs with the onset of darkness when oxidative stress and UV dam age would be lower than during the day, but cell division is not Functional impact of the circadian cycle on the prokaryotic cell Many efforts have been undertaken to decipher the biochemical basis of the oscillatory machinery and the pathways it controls. However, most approaches have been based on genetics, as it is not easy to predict and model the biochemical processes for such a complex mechanism. Thus, mainly mutants that target a compo nent gene of the clock machinery have been analyzed to infer their possible roles and their involvement in the circadian clock. Most of the knowledge about the cellular processes that the circadian clock gates comes from alterations in such cellular functional pathways as result of the mutation of one clock component or by determining the rhythmicity of cellular processes. Conservation of motifs and residues in the KaiC protein of prokaryotes Considering that the biochemistry of the KaiC protein depends on conserved motifs (Walker motifs) for its activity as ATPase and some conserved residues for its autokinase activity, we searched for the presence of these motifs in other prokaryotic organisms, and specifically in L. pneumophila. We selected those prokaryotes that contained both, kaiB and kaiC sequences including L. pneu mophila KaiC (Table 1) and aligned the protein sequence using the program ClustalX v.2.0.12 (Larkin et al., 2007) (Figure 2). Interestingly, all prokaryotic KaiC sequences aligned contained a well conserved Walker motif (GxxxxGK(T/S)) in the first KaiC domain (Figure 2). It has been reported that in bacteria the KaiC proteins usually contain a double KaiC domain but in some archea shorter KaiC sequences containing only a single domain can occur (Dvornyk et al., 2003), as also shown in Figure 2. Those organisms that have both KaiC domains show also a well conserved second Walker motif, but L. pneumophila and Flavobacterium johnsoniae exhibit a variant in the motif (GxxxxAK(T/S)). Furthermore, in most of the KaiC protein sequences, the potential N-terminal phosphorylation sites are well conserved (T426, S431, and T432 in S. elongatus). However, the T426 residue is also missing in F. johnsoniae and three δ-proteobacteria (Figure 2). The S431 phosphorylation residue is not conserved in Allochromatium vino sum and the T432 residue is replaced by an S residue in L. pneu mophila, and others like Geobacter sp., Rhodobacter sphaeroides, and Spirosoma linguale but that it may still be a phosphorylation target residue (Figure 2). Frontiers in Microbiology \| Cellular and Infection Microbiology Evolution of the circadian oscillator proteins in organisms containing KaiABC systems vs KaiBC systems An example of the kaiBC system is the circadian- like rhythm present in the purple bacterium Rh. sphaeroides (Min et al., 2005). and the few differences involved only the positions of bacteria not belonging to any of the main taxonomic groups (classified as “Others” in Figure 3). Phylogenetic analysis of the KaiC sequences, gave also very similar topologies with both methods although not identical. The main difference was that in the likelihood tree (Figure 3), the clade containing Legionella, some Proteobacteria and Bacteroidetes grouped together with the clade containing among others Cyanobacteria, however in the distance tree the mentioned clade grouped with the rest of proteobacteria. Whereas in the likeli hood tree the grouping node was supported by a bootstrap value of 79, there was no bootstrap value supporting this node in the distance tree. Thus we refer in our analysis to the likelihood tree shown in Figure 3. Although, circadian systems might also be present in other prokaryotes, the kai sequences in these prokaryotes might also have evolved functional divergence/convergence at least in those prokaryotes that do not show signs of lateral transfer. To get fur ther insight into the evolution of the KaiC proteins we have under taken an updated phylogenetic analysis of the KaiC sequence of all organisms listed in Table 1. In order to have a phylogenetic marker allowing to infer possible horizontal gene transfer, we also retrieved the RpoB sequence for the selected organisms from the NCBI database. As expected the phylogeny based on RpoB allowed good phylogenetic resolution comparable to the one obtained with the 16S rRNA. However, the advantage is, that rpoB is a single copy gene and thus no intra-genomic heterogeneity among copies as seen for the 16S rRNA is present. Both groups of sequences were aligned using the program ClustalX v.2.0.9 (Larkin et al., 2007). The best evolutionary model was selected using Prottest (Abascal et al., 2005). Using the best model accord ing to AIC criterion, phylogenetic trees were constructed using both a distance method (Neighbor-joining) and likelihood with 300 replicates of bootstrap. Distance methods were run using MEGA4 (Tamura et al., 2007) with the option “Pairwise deletion” in the case of RpoB and the likelihood tree was obtained by using Phyml (Guindon et al., 2005). Evolution of the circadian oscillator proteins in organisms containing KaiABC systems vs KaiBC systems In S. elongates, mutations in any of the kai genes lead to arrhyth mic patterns of the luciferase gene driven by different promoters (Ishiura et al., 1998). However, circadian clock systems exist also in organisms that do not have the kaiA gene like P. marinus and Rh. sphaeroides. With respect to the evolutionary analysis of the circadian components in cyanobacteria it was logical to infer that in cyanobacteria more than one circadian system might exist. First evidence came from the finding that some cyanobacteria lack the kaiA gene (Dvornyk et al., 2003), one of the three circadian core proteins in S. elongatus PCC7942 (Ishiura et al., 1998) but still have circadian rhythms. Based on these observations, Dvornyk and colleagues proposed that different circadian systems might exist in bacteria. The kaiABC system that has been extensively studied in S. elongatus PCC7942, and the kaiBC system that evolved Frontiers in Microbiology \| Cellular and Infection Microbiology December 2010 \| Volume 1 \| Article 130 \| 6 Circadian clock genes in prokaryotes Loza-Correa et al. Figure 2 \| Alignment of the KaiC sequences from prokaryotes containing both kaiB and kaiC sequences in their genome. Walker motifs and phosphorylation residues are indicated. The numbers below indicate the amino acid position with respect to the full protein sequence. Parentheses below the alignment indicate the location of each motif or residue: CI (first KaiC domain), CII (second KaiC domain), and N-End (N-terminus of the protein). Figure 2 \| Alignment of the KaiC sequences from prokaryotes containing both kaiB and kaiC sequences in their genome. Walker motifs and phosphorylation residues are indicated. The numbers below indicate the amino acid position with respect to the full protein sequence. Parentheses below the alignment indicate the location of each motif or residue: CI (first KaiC domain), CII (second KaiC domain), and N-End (N-terminus of the protein). Figure 2 \| Alignment of the KaiC sequences from prokaryotes containing both kaiB and kaiC sequences in their genome. Walker motifs and phosphorylation residues are indicated. The numbers below indicate the amino acid position with respect to the full protein sequence. Parentheses below the alignment indicate the location of each motif or residue: CI (first KaiC domain), CII (second KaiC domain), and N-End (N-terminus of the protein). probably from the kaiABC through the loss of kaiA and other components of the input or output circadian pathways like cikA (Baca et al., 2010). www.frontiersin.org Evolution of the circadian oscillator proteins in organisms containing KaiABC systems vs KaiBC systems As shown in Figure 3, the RpoB tree allowed to clearly separate the different taxonomic groups and was supported by high bootstrap values in both, distance and likelihood trees. Both methods gave very similar topologies Interestingly, the organisms containing both the kaiB and kaiC genes grouped in two main clades: one containing cyanobacteria and some α-Proteobacteria and a second clade containing two subclades of Proteobacteria and Bacteroidetes. In contrast, most of the organisms that do not contain the kaiB gene sequence form another clade. Thus our phylogenetic analysis grouped organisms containing the putative KaiABC or KaiBC systems in prokaryotes in a coherent way (Figure 4). Although the KaiC tree groups the main taxonomical groups (Cyanobacteria, and Proteobacteria) in separated clades, numerous non-correspondences with respect to the RpoB tree topology are indicating possible multiple hori zontal transfer events in the evolution of this gene as previously suggested (Dvornyk et al., 2003). Some clear evidence of horizon tal transfer events well supported are the ones from proteobacte ria to the archea Halalkalicoccus jeotgali or from proteobacteria to the Ktedonobacteria Ktedonobacter racemifer. In contrast for L. pneumophila we cannot infer clear evidence of horizontal gene December 2010 \| Volume 1 \| Article 130 \| 7 www.frontiersin.org Circadian clock genes in prokaryotes Loza-Correa et al. Figure 3 \| Phylogenetic tree of rpoB and kaiC sequences of prokaryotes containing kaiB and kaiC in their genome. (A) RpoB amino acid sequence tree obtained by the neighbor-joining method. (B) KaiC amino acid sequences obtained by the likelihood method. The bootstrap values presented at corresponding nodes were obtained from 300 replicates. Values under 50 are not indicated. transfer since Legionella is grouping with other Proteobacteria in the same clade. However the fact that this clade groups with Cyanobacteria and less with other proteobacteria may indicate either a lack of phylogenetic resolution of this gene or a differe origin for KaiC in different Proteobacteria resulting from horizon transfer or convergent evolution. of rpoB and kaiC sequences of prokaryotes containing kaiB and kaiC in their genome. (A) RpoB amino acid sequence tree method. (B) KaiC amino acid sequences obtained by the likelihood method. The bootstrap values presented at corresponding nodes es. Values under 50 are not indicated. Figure 3 \| Phylogenetic tree of rpoB and kaiC sequences of prokaryotes containing kaiB and kaiC in their genome. (A) RpoB amino acid sequence tree obtained by the neighbor-joining method. Evolution of the circadian oscillator proteins in organisms containing KaiABC systems vs KaiBC systems (B) KaiC amino acid sequences obtained by the likelihood method. The bootstrap values presented at corresponding nodes were obtained from 300 replicates. Values under 50 are not indicated. transfer since Legionella is grouping with other Proteobacteria in the same clade. However the fact that this clade groups with Cyanobacteria and less with other proteobacteria may indicate either a lack of phylogenetic resolution of this gene or a different origin for KaiC in different Proteobacteria resulting from horizontal transfer or convergent evolution. December 2010 \| Volume 1 \| Article 130 \| 8 Frontiers in Microbiology \| Cellular and Infection Microbiology Circadian clock genes in prokaryotes Loza-Correa et al. Figure 4 \| Maximum likelihood unrooted KaiC amino acid sequence tree. Organisms containing both kaiB and kaiC systems are grouped in two main clades: Cyanobacteria and α-Proteobacteria (green), and Proteobacteria and Bacteroidetes (pink). In these groups some organisms that do not belong to the main phylogenetic group are present. This grouping indicates potential events of lateral gene transfer. Figure 4 \| Maximum likelihood unrooted KaiC amino acid sequence tree. Organisms containing both kaiB and kaiC systems are grouped in two main clades: Cyanobacteria and α-Proteobacteria (green), and Proteobacteria and Bacteroidetes (pink). In these groups some organisms that do not belong to the main phylogenetic group are present. This grouping indicates potential events of lateral gene transfer. References mophila genome for exploitation of host cell functions and high genome plasticity. Nat. Genet. 36, 1165–1173. Johnson, C. H., Egli, M., and Stewart, P. L. (2008). Structural insights into a circa- dian oscillator. Science 322, 697–701. web server for fast maximum likeli- hood-based phylogenetic inference. Nucleic Acids Res. 33, W557– W559. web server for fast maximum likeli- hood-based phylogenetic inference. Nucleic Acids Res. 33, W557– W559. web server for fast maximum likeli- hood-based phylogenetic inference. 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W., van Oudenaarden, A., and Golden, S. S. (2010). Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elon- gatus. References Cell 140, 529–539. Katayama, M., Kondo, T., Xiong, J., and Golden, S. S. (2003). ldpA encodes an iron-sulfur protein involved in light- dependent modulation of the circa- dian period in the cyanobacterium Synechococcus elongatus PCC 7942. J. Bacteriol. 185, 1415–1422. Axmann, I. M., Duhring, U., Seeliger, L., Arnold, A., Vanselow, J. T., Kramer, A., and Wilde, A. (2009). Biochemical evidence for a timing mechanism in prochlorococcus. J. Bacteriol. 191, 5342–5347. Ito, H., Kageyama, H., Mutsuda, M., Nakajima, M., Oyama, T., and Kondo, T. (2007). Autonomous synchroniza- tion of the circadian KaiC phosphor- ylation rhythm. Nat. Struct. Mol. Biol. 14, 1084–1088. Dvornyk, V., and Nevo, E. (2003). Genetic polymorphism of cyanobacteria under permanent natural stress: a lesson from the “Evolution Canyons”. Res. Microbiol. 154, 79–84. Kim, Y. I., Dong, G., Carruthers, C. W. Jr., Golden, S. S., and LiWang, A. (2008). The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria. Proc. Natl. Acad. Sci. U.S.A. 105, 12825–12830. Dvornyk, V., Vinogradova, O., and Nevo, E. (2003). Origin and evolution of circa- dian clock genes in prokaryotes. Proc. Natl. Acad. Sci. U.S.A. 100, 2495–2500. Baca, I., Sprockett, D., and Dvornyk, V. (2010). Circadian input kinases and their homologs in Cyanobacteria: evo- lutionary constraints versus architec- tural diversification. J. Mol. Evol. 70, 453–465. Ivleva, N. B., Bramlett, M. R., Lindahl, P. A., and Golden, S. S. (2005). LdpA: a component of the circadian clock senses redox state of the cell. EMBO J. 24, 1202–1210. Kitayama, Y., Iwasaki, H., Nishiwaki, T., and Kondo, T. (2003). KaiB func- tions as an attenuator of KaiC phos- phorylation in the cyanobacterial circadian clock system. EMBO J. 22, 2127–2134. Garcia-Pichel, F. (1998). Solar ultravio- let and the evolutionary history of cyanobacteria. Orig. Life Evol. Biosph. 28, 321–347. Ivleva, N. B., Gao, T., LiWang, A. C., and Golden, S. S. (2006). Quinone sens- ing by the circadian input kinase of the cyanobacterial circadian clock. Proc. Natl. Acad. Sci. U.S.A. 103, 17468–17473. Cazalet, C., Gomez-Valero, L., Rusniok, C., Lomma, M., Dervins-Ravault, D., Newton, H. J., Sansom, F. M., Jarraud, S., Zidane, N., Ma, L., Bouchier, C., Etienne, J., Hartland, E. L., and Buchrieser, C. (2010). Analysis of the Legionella longbeachae genome and transcriptome uncovers unique strategies to cause Legionnaires’ dis- ease. PLoS Genet. 6, e1000851. doi: 10.1371/journal.pgen.1000851. Acknowledgments h k d fi This work received financial support from the Institut Pasteur and the Centre National de la Recherche (CNRS). Maria Loza-Correa is holder of a fellowship from the Ministère délégué à l’enseignement supérieur et à la recherche (France) and Laura Gomez-Valero is holder of a postdoctoral fellowship from the Fondation pour la Recherche Médicale (FRM). Johnson, C. H., Egli, M., and Stewart, P. L. (2008). Structural insights into a circa- dian oscillator. Science 322, 697–701. mophila genome for exploitation of host cell functions and high genome plasticity. Nat. Genet. 36, 1165–1173. Conclusions in response to changing environments. The massive acquisition of genome sequence of the most diverse organisms in the last years has now identified many additional prokaryotic and archeal genomes that contain homologues of kai genes. Although the model circadian clock is composed of the kaiABC genes, only some cyanobacteria encode KaiA, which is also not present in the rest of the prokaryotes containing the kaiB and/or kaiC genes. However, recently it was shown that the cyanobacterium Prochlorococcus sp. Circadian rhythms are daily cycles of metabolic activity, gene expression, and other biological processes that are regulated by self- sustained intracellular oscillators. Cyanobacteria are among the oldest organisms on earth and they are very successful in ecological plasticity and adaptability. Cyanobacteria were the first prokaryo tes reported to have the circadian clock regulated by three genes, kaiA, kaiB, and kaiC allowing adaptation and enhancing fitness December 2010 \| Volume 1 \| Article 130 \| 9 www.frontiersin.org Circadian clock genes in prokaryotes Loza-Correa et al. strain MED4 lacking kaiA displays the same biochemical func- tions than those from S. elongatus, having a KaiABC system. It might be expected that an evolutive adjustment of mechanistical features of the cyclic process occurred due to the absence of kaiA. Indeed Axmann et al. (2009) have shown that there is a differ- ence in the effect that KaiB exerts on KaiC dephosphorylation in Prochlorococcus sp. strain MED4. Thus, also in other bacteria tim- ing mechanism might exist that are regulated by the kaiBC system. It will be challenging to investigate the function of the diverse Kai homologues present in prokaryotes and archea. We are particularly interested in the KaiBC proteins encoded by L. pneumophila an environmental organism that also inhabits natural fresh waters like cyanobacteria. Thus L. pneumophila is also exposed to a series of photochemical reactions in water that could lead to the produc- tion of reactive oxygen species. Furthermore, L. pneumophila is an intracellular pathogen that parasitizes fresh water protozoa and that can also infect human alveolar macrophages causing a sever pneumonia. 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https://openalex.org/W2490553033	https://obzornik.zbornica-zveza.si/index.php/ObzorZdravNeg/article/download/93/90	Slovene	null	Izkušnje žensk ob nenačrtovanem porodu zunaj porodnišnice	Obzornik zdravstvene nege	2,016	cc-by	7,145	Izvirni znanstveni članek/Original scientific article Izvirni znanstveni članek/Original scientific article IZVLEČEK IZVLEČEK Uvod: Porodi zunaj porodnišnice so v Sloveniji redkost. Nekaj teh porodov se zgodi tudi v Zgornjem Posočju, ki je posebno zaradi svojega geografskega položaja in časovne oddaljenosti do najbližje porodnišnice. Cilj raziskave je bil pridobiti poglobljen vpogled v doživljajski svet porodnic, ki so nenačrtovan porod zunaj porodnišnice izkusile, in zaznati oceno kakovosti ponujene obporodne skrbi skozi občuteno zadovoljstvo. Metode: Raziskava je temeljila na kvalitativni metodologiji. V namenski vzorec je bilo vključenih 10 porodnic, ki so rodile med letoma 2004 in 2014. Podatki so bili pridobljeni s pomočjo delno strukturiranih intervjujev v juniju 2014 in analizirani z metodo analize besedila. Rezultati: Z metodo analize besedila je bilo identificiranih pet tem, in sicer (1) proces rojevanja – rituali, prakse in doživljanja, (2) podoživljanje poroda, (3) v žensko osredotočena skrb, (4) zaznana stopnja zadovoljstva z institucionalno obravnavo in (5) zaznavanje poroda med nosečnostjo – želje in pričakovanja. Diskusija in zaključek: Ugotovitve nakazujejo, da so med porodom v ospredju ljudje, ki so ob porodnici, in njihov odnos do nje. Čeprav se porodnice takrat morda res ne zavedajo vseh morebitnih tveganj, se tega zavedajo zdravstveni delavci ob njej. Vključene v raziskavo so svojo porodno izkušnjo izkusile kot pozitivno in obenem izrazile zadovoljstvo do obporodne zdravstvene oskrbe. Nadaljnje raziskovanje bi moralo vključevati vidik zdravstvenih delavcev. Ključne besede: Zgornje Posočje; oddaljenost; babica; medosebni odnos; opolnomočenje Uvod: Porodi zunaj porodnišnice so v Sloveniji redkost. Nekaj teh porodov se zgodi tudi v Zgornjem Posočju, ki je posebno zaradi svojega geografskega položaja in časovne oddaljenosti do najbližje porodnišnice. Cilj raziskave je bil pridobiti poglobljen vpogled v doživljajski svet porodnic, ki so nenačrtovan porod zunaj porodnišnice izkusile, in zaznati oceno kakovosti ponujene obporodne skrbi skozi občuteno zadovoljstvo. Metode: Raziskava je temeljila na kvalitativni metodologiji. V namenski vzorec je bilo vključenih 10 porodnic, ki so rodile med letoma 2004 in 2014. Podatki so bili pridobljeni s pomočjo delno strukturiranih intervjujev v juniju 2014 in analizirani z metodo analize besedila.i Key words: Upper Soča region; distance; midwife; interpersonal relationship; empowerment j j Metode: Raziskava je temeljila na kvalitativni metodologiji. V namenski vzorec je bilo vključenih 10 porodnic, ki so rodile med letoma 2004 in 2014. Podatki so bili pridobljeni s pomočjo delno strukturiranih intervjujev v juniju 2014 in analizirani z metodo analize besedila. IZVLEČEK Rezultati: Z metodo analize besedila je bilo identificiranih pet tem, in sicer (1) proces rojevanja – rituali, prakse in doživljanja, (2) podoživljanje poroda, (3) v žensko osredotočena skrb, (4) zaznana stopnja zadovoljstva z institucionalno obravnavo in (5) zaznavanje poroda med nosečnostjo – želje in pričakovanja. Ksenija Kragelj, dipl. m. s.; Zdravstveni dom Tolmin, Prešernova ul. 6, 5220 Tolmin Kontaktni e-naslov/ Correspondence e-mail: kkragelj@gmail.com viš. pred. mag. Mirko Prosen, dipl. zn., univ. dipl. org.; Univerza na Primorskem, Fakulteta za vede o zdravju, Polje 42, 6310 Izola Ksenija Kragelj, dipl. m. s.; Zdravstveni dom Tolmin, Prešernova ul. 6, 5220 Tolmin Diskusija in zaključek: Ugotovitve nakazujejo, da so med porodom v ospredju ljudje, ki so ob porodnici, in njihov odnos do nje. Čeprav se porodnice takrat morda res ne zavedajo vseh morebitnih tveganj, se tega zavedajo zdravstveni delavci ob njej. Vključene v raziskavo so svojo porodno izkušnjo izkusile kot pozitivno in obenem izrazile zadovoljstvo do obporodne zdravstvene oskrbe. Nadaljnje raziskovanje bi moralo vključevati vidik zdravstvenih delavcev. Kontaktni e-naslov/ Correspondence e-mail: kkragelj@gmail.com viš. pred. mag. Mirko Prosen, dipl. zn., univ. dipl. org.; Univerza na Primorskem, Fakulteta za vede o zdravju, Polje 42, 6310 Izola Članek je nastal na osnovi diplomskega dela Ksenije Kragelj: Porodi izven bolnišnice: kvalitativna analiza doživljanja žensk v Zgornjem Posočju (2014). Prejeto/Received: 8. 2. 2016 Sprejeto/Accepted: 31. 5. 2016 ABSTRACT Introduction: Out-of-hospital births are rare in Slovenia. Some of these births occur also in the Upper Soča region, mainly due to its geographical location and the travel time to the nearest hospital, specifically. The aim of this study was to obtain a deeper insight into childbearing women's experience of unplanned out-of- hospital birth, and to evaluate the satisfaction with the quality of perinatal care provided. Methods: The study was based on a qualitative methodology. The purposive sample included 10 pregnant women who gave birth between 2004 and 2014. The data were obtained through semi-structured interviews in June 2014 and were analysed by using the method of content analysis. Results: The analysis yields five thematic clusters, namely (1) birth process – rituals, practices and experiences, (2) re-experiencing birth, (3) woman-centred care, (4) perceived level of satisfaction with institutional treatment, and (5) feelings, desires and perceptions of women during pregnancy which are related to birth. Discussion and conclusion: The findings suggest that during unplanned out-of-hospital birth, the most important is the active and passive support of persons present and their attitude towards the woman in labour. Although women are not fully aware of all potential risks, the health professional present should be prepared and take all the preventive measures. The study participants perceived home birth as a positive Results: The analysis yields five thematic clusters, namely (1) birth process – rituals, practices and experiences, (2) re-experiencing birth, (3) woman-centred care, (4) perceived level of satisfaction with institutional treatment, and (5) feelings, desires and perceptions of women during pregnancy which are related to birth.hi (2) re-experiencing birth, (3) woman-centred care, (4) perceived level of satisfaction with institutional treatment, and (5) feelings, desires and perceptions of women during pregnancy which are related to birth. Discussion and conclusion: The findings suggest that during unplanned out-of-hospital birth, the most important is the active and passive support of persons present and their attitude towards the woman in labour. Although women are not fully aware of all potential risks, the health professional present should be prepared and take all the preventive measures. The study participants perceived home birth as a positive experience and also expressed high level of satisfaction with perinatal health care. Further research should be undertaken to investigate into the attitudes of health care professionals' assisting in home birth. Discussion and conclusion: The findings suggest that during unplanned out-of-hospital birth, in labour. Izkušnje žensk ob nenačrtovanem porodu zunaj porodnišnice Women's experience of unplanned out-of-hospital birth Ksenija Kragelj, Mirko Prosen 2016. Obzornik zdravstvene nege, 50(2), pp. 135–143. 2016. Obzornik zdravstvene nege, 50(2), pp. 135–143. Uvod razumevanje tveganj, povezanih s takšnim porodom, in vprašanj o varnosti, kulturno normativna pričakovanja, osebna prepričanja, predhodne porodne izkušnje ter vidik pomembnih drugih in zdravstvenega osebja, pa nenačrtovan porod izven porodnišnice opredeljujejo druge značilnosti. Nenačrtovan porod zunaj porodnišnice je mogoče pričakovati pri ženskah, ki zanemarijo začetek poroda oziroma ga ne prepoznajo dovolj hitro, in v primerih hitrega poteka poroda; pri mnogorodnicah; pri veliki oddaljenosti do najbližje porodnišnice; pri prisotnih psihičnih težavah ali jezikovnih ovirah (Blondel, et al., 2011; Erlandsson, et al., 2015). Nenačrtovani porodi zunaj porodnišnice so pogostejši pri porodnicah iz nižjih družbenih slojev, medtem ko so načrtovani porodi pogostejši pri porodnicah iz višjih družbenih razredov (Blondel, et al., 2011), kavkaške rase, nekadilkah, starejših in iz socialno bolj privilegiranih okolij ter tudi mnogorodnicah (Serdinšek & Takač, 2016). Kot še ugotavljajo Blondel in sodelavci (2011), je v številnih državah opaziti dolgoročni trend zapiranja manjših porodnišnic, zlasti na tistih predelih, kjer se čas potovanja do naslednje porodnišnice zaradi zaprtja ni drastično povečal. Kljub temu avtorji izpostavljajo, da se bo delež nenačrtovanih porodov zunaj porodnišnice v prihodnje povečeval tudi zaradi tega razloga, še posebej v najbolj odročnih krajih. Nosečnost in porod sta naravna in enostavna dogodka v življenju ženske, a se hkrati lahko spremenita v zelo zapletena (Njenjić & Skela Savič, 2011; Spaich, et al., 2013), še posebej je to v takšnih primerih določujoče v okolju, ko zaradi oddaljenosti do najbližje porodnišnice ni mogoče v celoti zagotoviti takšne zdravstvene oskrbe, kot bi jo v porodnišnici. Med letoma 2002 in 2011 je bilo v Sloveniji 234 porodov zunaj porodnišnice. Gre za redke dogodke (0,12 % vseh porodov oziroma dva na mesec), pri čemer so lahko letna nihanja naključna (Zupan, 2013). Del teh se zgodi tudi na enem od najbolj obsežnih terenov v severozahodni Sloveniji, to je v Zgornjem Posočju, ki obsega Bovško, Kobariško in Tolminsko. Vsi tisti, ki ne poznajo razgibanega hribovskega terena Zgornjega Posočja, si težje predstavljajo čas, ki ga potrebujejo porodnice s tega območja, da varno pridejo do najbližje porodnišnice. Zaradi omenjenega so na tem območju vsako leto od dva do štirje porodi zunaj porodnišnice (Kragelj, 2014), kar vpliva na organizacijo ponujene zdravstvene oskrbe (Kragelj & Prosen, 2015). Ob tem nikakor ni nezanemarljivo doživljanje poroda pri porodnici. Ugotovitve namreč kažejo, da lahko ženske, ki rodijo nenačrtovano zunaj porodnišnice in pričakujejo porod v institucionalnem okolju, izkusijo občutke ranljivosti in stresa (Scott & Esen, 2005; Spaich, et al., 2013; Erlandsson, et al., 2015). ABSTRACT Although women are not fully aware of all potential risks, the health professional present should be prepared and take all the preventive measures. The study participants perceived home birth as a positive experience and also expressed high level of satisfaction with perinatal health care. Further research should be undertaken to investigate into the attitudes of health care professionals' assisting in home birth. http://dx.doi.org/10.14528/snr.2016.50.2.93 Kragelj, K. & Prosen, M., 2016. / Obzornik zdravstvene nege, 50(2), pp. 135–143. 136 Namen in cilji / Obzornik zdravstvene nege, 50(2), pp. 135–143. 137 Opis poteka raziskave in obdelave podatkov Z uporabo principa snežne kepe je bilo povabilo za sodelovanje v raziskavi posredovano nekaterim ženskam, ki so rodile med letoma 2004 in 2014 v Zgornjem Posočju ter ohranile stik s prvo avtorico, ki je bila v nekaterih primerih zaradi svojih izkušenj kot medicinska sestra − babica prisotna ob porodu. Ta jih je obvestila o raziskovalni nameri in jih prosila, naj o tem obvestijo tudi druge, ki jih poznajo. Potencialne intervjuvanke, ki so se odzvale povabilu za sodelovanje, so bile seznanjene z namenom, cilji in potekom raziskave. S tistimi, ki so potrdile sodelovanje, smo se dogovorili za termin in kraj srečanja po njihovem izboru, ob pogoju, da bosta omogočeni zasebnost in intimnost. Intervjuji so potekali junija 2014, pri čemer smo sledili etičnim smernicam družboslovnega raziskovanja, ki jih povzema Bulmer (2008), kar je zaradi vpletenosti prve avtorice med drugim pomenilo tudi ohranjati receptivno držo in načela Helsinško- Tokijske deklaracije. Pridobili smo dovoljenje za opravljanje raziskave od delovne organizacije (Zdravstveni dom Tolmin). Pred začetkom intervjuja so bile intervjuvanke ponovno seznanjene z namenom in cilji raziskave, ukrepi za zagotavljanje anonimnosti, zaupnosti oziroma varovanjem osebnih podatkov in možnostjo, da se na njihovo željo kadarkoli prekine intervju. Omenjeni elementi so sestavljali informirano soglasje, ki so ga morale intervjuvanke, ki so se odločile za prostovoljno sodelovanje, podpisati. Intervjuji so bili zvočno snemani. Posamezni intervju je v povprečju trajal 40 minut. Ob koncu vseh intervjujev so bili narejeni njihovi transkripti. Z namenom varovanja anonimnosti so bila imena sodelujočih nadomeščena z namišljenimi imeni. Imena drugih oseb ali krajev so bila v besedilu izpuščena in izpust ustrezno označen. Nasičenost podatkov (Fusch & Ness, 2015) se je nakazovala pri devetem intervjuju, ko med povedanim ni bilo zaznati nobenih novih dejstev, in je bila pozneje potrjena v fazi kodiranja. Opis instrumenta Podatki so bili pridobljeni s pomočjo delno strukturiranih intervjujev. Pri tej obliki si raziskovalec pred intervjujem pripravi glavna vprašanja, ki jih zastavlja vsakemu intervjuvancu posebej. Podvprašanja, če so potrebna, pa oblikuje in postavlja med intervjujem (DiCicco-Bloom & Crabtree, 2006). Pred začetkom intervjuja so intervjuvanke povedale nekaj osnovnih demografskih podatkov (starost ob porodu, dosežena stopnja izobrazbe, oddaljenost do najbližje bolnišnice, zaporedni porod). Osrednji del intervjuja so bila vprašanja, povezana z izkušnjo začetka poroda (npr. kako se je porod začel, ste odlašali z odhodom v porodnišnico, ste razmišljali o porodu zunaj porodnišnice), porodom (npr. kako ste doživeli in sprejeli dejstvo, da porod v porodnišnici ne bo mogoč, koliko ste zaupali zdravstvenemu osebju) in časom po porodu (npr. kako ste doživljali trenutke po rojstvu, kako ste bili sprejeti v porodnišnici). Namen in cilji Pri porodih zunaj porodnišnice ali porodih na domu ločimo nenačrtovane in načrtovane porode na domu, te pa lahko razdelimo na tiste, pri katerih je prisoten spremljevalec ali pa ne (Serdinšek & Takač, 2016). Čeprav so nenačrtovani porodi zunaj porodnišnice naključni, niso nepredvidljivi in zagotovo se jih vsako leto nekaj tudi zgodi (McLelland, et al., 2013). McLelland in sodelavci (2013) ob pregledu literature ugotavljajo, da je definicija nenačrtovanega poroda zunaj porodnišnice težavna, saj si različni avtorji pomen nenačrtovanega poroda razlagajo drugače. Tako nekateri ta pojem opisujejo kot nepričakovan porod novorojenca pred prihodom babice ob načrtovanem porodu na domu, spet drugi pa kot porod na neprimerni lokaciji brez strokovne pomoči. Najpogosteje uporabljeno definicijo nenačrtovanega poroda zunaj porodnišnice navajata Scott in Esen (2005), ki pravita, da je nenačrtovan porod zunaj porodnišnice porod, ki je bil načrtovan, da bo potekal v porodnišnici, vendar se je zgodil pred prihodom v porodnišnico. Takšni porodi se zgodijo nepričakovano, zato lahko potekajo tudi brez strokovne pomoči, pogosto tudi na poti v porodnišnico. Erlandsson in sodelavci (2015) opredeljujejo porod zunaj porodnišnice kot tisti porod, ki se zgodi zunaj porodnišnice ne glede na kraj zaključka tretje porodne dobe. Porod je za vsako žensko zelo osebna in čustvena življenjska izkušnja, ki simbolizira prehod v materinstvo. Mnenja o tem, ali porod sodi v porodnišnico ali v drugo okolje zunaj porodnišnice po izbiri porodnice, so med profesionalci in laično javnostjo deljena. Namen članka ni posegati v tovrstne polemike, temveč predstaviti doživljanje rojevanja v okolju, ki je zaradi svojih reliefnih in podnebnih značilnosti lahko povsem nedostopno za bolnišnično oskrbo tudi v primerih, ko je resno ogroženo zdravje matere in ploda oziroma novorojenca. Boljše razumevanje porodnic, ki nenačrtovano rodijo zunaj porodnišnice, omogoča prilagajanje ukrepov, ki v okolju, kjer takšni porodi niso redkost, prispevajo k zagotavljanju kakovostne zdravstvene oskrbe in posledično k ustvarjanju pozitivne porodne izkušnje. S tem namenom in ob dejstvu, da pregled literature kaže na pomanjkanje tovrstnih raziskav, je bil cilj raziskave pridobiti poglobljen vpogled v doživljanje porodnic, ki nenačrtovano rodijo zunaj porodnišnice. Oblikovani sta bili naslednji raziskovalni vprašanji: Oblikovani sta bili naslednji raziskovalni vprašanji: Kako porodnice doživljajo nenačrtovan porod (ob − prisotnem zdravstvenem osebju) zunaj porodnišnice? Kateri vidiki doživljajskega sveta (osebni, družbeni, − zdravstveni ipd.) porodnic so v ospredju pri nenačrtovanem porodu (ob prisotnem zdravstvenem osebju) zunaj porodnišnice? Coxon in sodelavci (2015) pravijo, da na načrtovan porod zunaj porodnišnice vplivajo dejavniki, kot so Kragelj, K. & Prosen, M., 2016. Metode Ob ugodnih vremenskih razmerah bi do prihoda v porodnišnico dve intervjuvanki potrebovali 45 minut, pet 60 minut in tri 90 minut. Za dosego zastavljenih ciljev, kjer je bil v ospredju proučevanja doživljajski svet porodnic, ki so nenačrtovano rodile zunaj porodnišnice in je bilo prisotno zdravstveno osebje, je bila izbrana kvalitativna paradigma, ki sledi interpretativnemu modelu. Kvalitativno raziskovanje namreč analizira konkretne primere v njihovi časovni in krajevni specifiki, pri čemer upošteva izkušnje in doživljanja ljudi v teh okoliščinah (Flick, 2002). Opis vzorca V namenski vzorec so bile vključene porodnice, ki so nenačrtovano rodile zunaj bolnišnice in kjer je bilo prisotno zdravstveno osebje. Za sodelovanje v raziskavi se je odločilo 10 porodnic, kar je z vidika velikosti vzorca, če upoštevamo izbrani raziskovalni dizajn, zadostno (Steen & Roberts, 2011). Kljub temu smo sledili kriteriju nasičenosti podatkov (Fusch & Ness, 2015). ) Dve intervjuvanki sta rodili leta 2004, ena leta 2005, po dve leta 2008, 2010 in 2011 ter ena leta 2013. Za eno od intervjuvank je bil to četrti zaporedni porod, za tri tretji, za pet drugi porod, ena pa je bila prvorodnica. Z vidika izobrazbene strukture sta imeli dve intervjuvanki končano osnovno šolo, dve poklicno, tri srednjo šolo, ena visoko šolo in dve univerzitetni študijski program. V osmih primerih je bil porod spontan, v dveh pa je bila zaradi zapletov potrebna epiziotomija. Povprečna starost intervjuvank je bila v času poroda 30,5 leta. Najmlajša intervjuvanka je takrat dopolnila 22 let, najstarejša pa 37 let. Glede na oddaljenost intervjuvank od najbližje bolnišnice je v povprečju najbližja porodnišnica oddaljena 40 kilometrov, najbolj oddaljena pa je 57 kilometrov stran. Podatki so bili analizirani z metodo analize besedila. Uporabljena je bila tematska analiza besedila, pri kateri gre za proces analize podatkov v skladu s podobnostmi, povezavami in razlikami med podatki (Gibson & Brown, 2009). Tematska analiza je potekala tako, da so bila besedila večkrat prebrana, kodirana in kode kategorizirane, ter na podlagi kategorij oblikovane teme (Steen & Roberts, 2011). Zaradi prisotnosti prve avtorice ob nekaterih nenačrtovanih porodih zunaj porodnišnice sta z namenom zagotavljanja zanesljivosti in kredibilnosti rezultatov analizo besedila avtorja opravljala ločeno. Analizo je najprej opravila prva avtorica, nato drugi avtor (Fusch & Ness, 2015), sledilo je usklajevanje diskrepanc obeh analiz, ki pa Kragelj, K. & Prosen, M., 2016. / Obzornik zdravstvene nege, 50(2), pp. 135–143. Opis vzorca 138 Tabela 1: Identificirane teme Table 1: Identified themes Tema/Theme Kategorija/Category Frekvenca kod/ Frequency code Proces rojevanja: rituali, prakse in doživljanja Nepričakovan začetek poroda Rituali pred odhodom v porodnišnico Nezavedanje o zapletih pri porodu Odsotnost strahu pred porodom zunaj bolnišnice Doživljanje poroda (pozitivno) Prvi stik matere z novorojencem Vpliv kulturnega okolja – partner Spremljajoča birokracija 108 Podoživljanje poroda Zadovoljstvo ob porodu zunaj bolnišnice Refleksija (podoživljanje poroda) 49 V žensko osredotočena skrb Medsebojno zaupanje Avtonomija 24 Zaznana stopnja zadovoljstva z institucionalno obravnavo Institucionalna obravnava med nosečnostjo in po porodu Medikalizacija 23 Zaznavanje poroda med nosečnostjo: želje in pričakovanja Želja po naravnem porodu Psihofizična pripravljenost na porod 11 bo potekal v porodnišnici, ni prestrašilo ali omajalo njihovega zaupanja v zdravstveno osebje. Bolj kot kraj rojevanja pa k ustvarjanju pozitivne porodne izkušnje pomembno prispeva zaupanje porodnice v zdravstveno osebje. so se v glavnini nanašale na ustrezno poimenovanje identificiranih tem. Pri predstavitvi rezultatov je bila upoštevana tudi frekvenca posameznih kod. Rezultati »V tistem trenutku mislim, da se nisem zavedala tveganja, ki ga tak porod prinaša. Ne, o nobenih komplikacijah med porodom nisem razmišljala, niti to, da ne meni ali mojemu novorojenčku zdravstveno osebje ne bo moglo pomagati. Jaz sem se počutila tako v varnih rokah in nisem nič razmišljala v povezavi s tem. Res ne.« (Tanja, 34 let) Identificiranih je bilo pet tem, ki opredeljujejo doživljajski svet porodnic, ki so izkusile porod zunaj porodnišnice, in sicer: (1) proces rojevanja: rituali, prakse in doživljanja, (2) podoživljanje poroda, (3) v žensko osredotočena skrb, (4) zaznana stopnja zadovoljstva z institucionalno obravnavo, (5) zaznavanje poroda med nosečnostjo: želje in pričakovanja (Tabela 1). »Ko sem prišla enkrat v zdravstveni dom, sem vedela, da je tam usposobljeno osebje. Zaupala sem babici in ostalim, ki so bili okrog mene. Takrat mene ni bilo nič več strah, celo odleglo mi je, zato so se popadki celo malo ustavili, potem pa so se zopet stopnjevali.« (Lucija, 37 let) Proces rojevanja: rituali, prakse in doživljanja Tu sem imela stalno enega ob sebi, zato nisi imel časa razmišljati o strahu ali pa o bolečini.« (Sara, 28 let) »Tu je bilo vse bolj sproščeno. To mi je bil tako dober občutek, tu je bilo vse bolj domače.« (Tanja, 34 let) j j j Devet intervjuvank, ko pogovor nanese na stvari, ki bi jih spremenile ob rojevanju, ugotavlja, da ne bi spremenilo ničesar. »Zelo lepo sem doživela ta porod. Bil je enkraten, že zaradi tega, ker je bil mož ves čas ob meni. Tu ste se pa vsi ves čas z mano pogovarjali in to je bilo tako krasno. Jaz sem to tako lepo doživljala [...] In smo rodili. To je bilo krasno. Najbolj lepo od vsega pa mi je bilo, ko sem po porodu dobila na trebuh ogreto bombažno plenico. To je bilo tako lepo, da bi bilo dobro, če bi to še kje prakticirali. To je bilo tako, kot če bi položili eno zelo nežno stvar na obolelo mesto. To zelo dobro dene. Jaz sem se počutila zelo dobro, pa še porod ni bil naporen.« (Lucija, 37 let) »[...] Čutila sem, da je porod že zelo, zelo pri koncu. V bistvu sem si takrat želela, da ostane vse tako, kot je. Rodila sem zelo hitro, v bistvu dva močna popadka in je bil moj novorojenček zunaj. Tudi danes bi ravnala popolnoma enako, če bi se znašla v taki situaciji. Za nazaj ne bi nič spreminjala. Vse je bilo super.« (Julija, 32 let) »Mož je bil malo živčen in nestrpen. Moje doživljanje tega poroda je malce drugačno. Tisti čas sem bolj razmišljala o mojih dveh otrocih, ki sta bila doma. Zavedala sem se, da je na poti še eden, in sem mislila, kako bomo vse to izpeljali. Na sam porod sploh nisem mislila. Moje misli so bile prav drugje. Babici sem rekla, da ne morem več in da bo dojenček prišel ven. Babica se je pripravila za porod in sem rodila. Vse je potekalo tako, kot je moralo. Za nazaj ne bi nič spreminjala. Lepo je bilo in nič drugače ne bi ravnala, tudi če bi se danes znašla v taki situaciji.« (Ivana, 27 let) Kot je razvidno, je k pozitivni izkušnji dodatno prispevala prisotnost zdravstvenega osebja in partnerja, čeprav ta v vseh primerih ni bil prisoten. Pozitivno izkušnjo je sooblikoval prvi stik matere z novorojencem. »Ja, takoj po porodu sem dobila novorojenčka na trebuh. Podoživljanje poroda »Zaupala sem babici in ostalim, ki so bili okrog mene. Popolnoma sem zaupala osebju, najbolj pa babici, zato ker je bila ona tista oseba, ki je bila najbolj v stiku z mano v tistem trenutku.« (Lucija, 37 let) Tema »podoživljanje poroda« je tesno povezana s prvo identificirano temo. Intervjuvanke priznavajo, da se velikokrat ozrejo nazaj in razmišljajo o porodu oziroma porodih. Analizirajo vsako podrobnost in jo poskušajo osvetliti z vseh strani. Pri tem največkrat uporabijo besedo »lepo je bilo«. Še posebej izstopajo v primerjavah poroda tiste intervjuvanke, ki so ga izkusile v porodnišnici in zunaj nje. »[…] Jaz ne morem nekomu reči, da mora roditi v porodnišnici, lahko se zgodi, da bo porod doma ali na poti v porodnišnico. To je njena odločitev.« (Ivana, 27 let) V žensko osredotočena skrb j j p j Tudi birokratizacija, ki spremlja porod zunaj porodnišnice, ni bistveno prispevala k zmanjšanju pozitivne porodne izkušnje, čeprav Mojca (27 let) omenja poznejše težave pri prijavi rojstva. Morda najpomembnejši del porodne izkušnje je za ženske individualizirana zdravstvena oskrba, ki upošteva njihove potrebe in želje. Pri njenem zagotavljanju izstopata medsebojno zaupanje, za katerega vse intervjuvanke pravijo, da so si ga zdravstveni delavci prislužili s profesionalnim pristopom, in avtonomija ženske, da samostojno odloča o svojem telesu, načinu in tudi kraju poroda. »[…] ni me motilo izpolnjevanje predpisanih obrazcev, saj sem že od prvega poroda vedela, da bom morala podpisati tista dva papirja. Vem, da se je zapletlo pri prijavi rojstva.« Proces rojevanja: rituali, prakse in doživljanja To je tako dober občutek. To je tako lepo. Več časa sem ga imela pri sebi kot prvič v porodnišnici. Res je bilo lepo, ko smo bili skupaj. Meni je bilo vse super. Ne vem, vse se mi je zdelo strokovno, istočasno pa tako domače. Res nisem nič pogrešala. [...] Tako kot sem vam že povedala, je bilo meni tako lepo, ničesar ni bilo, kar bi lahko rekla, da je manjkalo.« (Hana, 32 let) V primerjavi z drugimi intervjuvankami je Nina (37 let), ki ima za sabo več porodov, v svojem doživljanju poroda zunaj porodnišnice bolj pragmatična. »Če bi se danes ponovno znašla v taki situaciji, jaz sedaj težko razmišljam, kaj bi naredila, ker sem se znašla v taki situaciji, da sem potrebovala intenzivno nego po petem porodu kar nekaj časa in prav zaradi te izkušnje priporočam vsem, da rodijo v porodnišnici. Pri mojem četrtem porodu pa ne bi nič spreminjala (zunaj porodnišnice, op. a.).« (Nina, 37 let) »Takoj po porodu so mi novorojenčka položili na trebuh. Obrisali so ga, pokrili s toplo plenico in mi ga položili na trebuh. Ja, kar nekaj časa sem ga imela na trebuhu, potem smo se še dojili. Nič ni manjkalo, nič ni bilo manj. Krasno.« (Lucija, 37 let) »Prvega novorojenčka so takoj po rojstvu položili očetu v naročje, saj so se pripravljali še za rojstvo drugega. Vse je bilo hitro, vse je bilo lepo.« (Mateja, 29 let) Proces rojevanja: rituali, prakse in doživljanja Za večino intervjuvank je bil začetek poroda nepričakovan − bodisi zaradi njegovega hitrega stopnjevanja, oddaljenosti do pričakovanega datuma poroda ali lastne predstave o tem, kako naj bi se porod začel. Ob ugotovitvi intervjuvank, da se je porod začel, je mnoge od njih bremenilo dejstvo, da morajo kljub hitremu napredovanju poroda še pred odhodom od doma med drugim najprej poskrbeti za osebno higieno. Ko se je izkazalo, da porod v porodnišnici ne bo mogoč, se pri intervjuvankah niso pojavljali strahovi glede možnih zapletov, povezanih s porodom zunaj porodnišnice, ali dejstva, da pogoji na primarni ravni morda niso enaki tistim na sekundarni ali terciarni ravni zdravstvenega varstva, npr. v primeru oskrbe nedonošenega otroka. Tovrstni strahovi so se pojavili šele po porodu, ko so se skozi refleksijo ozirale nazaj. Mnogo bolj kot to je bilo med rojevanjem v ospredju medsebojno zaupanje med zdravstvenim osebjem in porodnico, zato jih dejstvo, da porod ne »Čisto umirjena sem bila po vaginalnem pregledu, ko mi je babica povedala, da ne bomo rodili v porodnišnici, ker sem jaz popolnoma zaupala babici. Čisto nič me ni bilo strah.« (Sara, 28 let) Vse porodnice, ki so rodile zunaj porodnišnice, so porod opredelile kot pozitivno porodno izkušnjo. »Moje doživljanje poroda je zelo lepo. Takoj, ko so me pripeljali skozi vrata v urgentno ambulanto, sem opazila, da se na radiatorju grejejo oblekice za dojenčka. Potem smo predihali popadke. Celotna popoldanska ekipa je ostala z mano in me bodrila. Močno sem zajela sapo in nato še enkrat in novorojenček je bil zunaj. Ja, ti popadki so bili zelo močni, veliko bolj kot cunami! V tistem trenutku je bilo vse super. Edino, kar mi je ostalo v spominu in mi gre na smeh še danes, je to, da babica ni imela v urgentni ambulanti CTG-ja in je poslušala utripe s tisto slušalko (Pinnardova slušalka, op. a.) in takrat smo morali biti tiho. To, da smo morali biti tiho, 139 Kragelj, K. & Prosen, M., 2016. / Obzornik zdravstvene nege, 50(2), pp. 135–143. to je bilo težko. Moje doživljanje poroda je zelo lepo. Tu sem imela stalno enega ob sebi, zato nisi imel časa razmišljati o strahu ali pa o bolečini.« (Sara, 28 let) »Tu je bilo vse bolj sproščeno. To mi je bil tako dober občutek, tu je bilo vse bolj domače.« (Tanja, 34 let) to je bilo težko. Moje doživljanje poroda je zelo lepo. Zaznana stopnja zadovoljstva z institucionalno obravnavo »Ja, spontano roditi izven porodnišnice je čisto drugače. Lepo je bilo. Na koncu je bilo okrog mene samo poznano zdravstveno osebje, vsi so me spodbujali, božali, držali za roko.« (Nina, 37 let) Sliko poroda pri večini intervjuvank sooblikuje tudi institucionalna obravnava med nosečnostjo in pozneje 140 Kragelj, K. & Prosen, M., 2016. / Obzornik zdravstvene nege, 50(2), pp. 135–143. nisem odločila. Ne, to pa ne.« (Hana, 32 let)i nisem odločila. Ne, to pa ne.« (Hana, 32 let)i po porodu, po premestitvi v porodnišnico. Temo opredeljujeta dve kategoriji, in sicer »institucionalna obravnava v času nosečnosti in po porodu« ter »medikalizacija«, ki se nanaša na prvo kategorijo, pri čemer nekatere to doživljajo pozitivno, nekatere pa negativno. Večina intervjuvank pozitivno opisuje institucionalno obravnavo v nosečnosti, medtem ko Mojca (27 let), ki je na dan predvidenega datuma poroda obiskala porodnišnico, v kateri je želela roditi, o tem pravi: Psihofizična priprava na porod je redko omenjena, še posebej pri prvorodnicah, kar da slutiti, da so v primerjavi z mnogorodnicami lahko slabše pripravljene na porod. Mojca (27 let), ki je že izkusila materinstvo, v tem pogledu izpostavlja zlasti psihično pripravo:i »Vse je v glavi, če se ti telesno in fizično pripraviš na tak porod, se ti ne more nič hudega zgoditi. Tudi doma lahko rodiš, samo pripravljen moraš biti na to.« (Mojca, 27 let) »Na termin poroda sem bila na pregledu v porodnišnici. Tam sem čakala za tista dva pregleda, ultrazvok in vaginalni pregled, cele štiri ure, skupaj z dveletnim sinčkom […] Res je bilo grozno, ker smo bile v čakalnici samo štiri nosečnice in smo tako dolgo čakale. Po vaginalnem pregledu so mi ponudili, da lahko ostanem v porodnišnici, ker sem že malo odprta. Odklonila sem. Šla sem domov.« Diskusija V Zgornjem Posočju je časovna oddaljenost do najbližje porodnišnice velika težava, še posebej za tiste porodnice, ki z odhodom odlašajo. Raziskava kaže, da se nekatere intervjuvanke, veliko bolj prvorodnice, ki so nenačrtovano rodile zunaj porodnišnice, niso zavedale pomembnosti časovne oddaljenosti in/ ali prepoznale znakov začetka poroda. Ugotovitve drugih raziskav kažejo, da je časovna oddaljenost do najbližje porodnišnice pomembno povezana z večjo pogostnostjo nenačrtovanega poroda (McLelland, et al., 2013) ter večjim deležem neonatalne mortalitete in morbiditete, ko govorimo o nenačrtovanem porodu (Scott & Esen, 2005; Blondel, et al., 2011; McLelland, et al., 2013; Gunnarsson, et al., 2014; Nguyen, et al., 2016). Čeprav je porod zunaj porodnišnice redek pojav tako pri nas kot v razvitem svetu (Blondel, et al., 2011; McLelland, et al., 2013; Gunnarsson, et al., 2014; Erlandsson, et al., 2015), je pomembno razumeti, kako ženske doživljajo takšen porod in kateri vidiki njenega doživljajskega sveta so v ospredju. Porod je univerzalen dogodek, vendar pa je njegovo doživljanje zelo individualno (Lindgren & Erlandsson, 2010), kar potrjuje tudi raziskava, in se spreminja s sociopolitičnim kontekstom, v katerega je umeščen (Records & Wilson, 2011), zato je analiziranje vsakdanjega življenja in porodnih pričevanj žensk nujna in legitimna sestavina vrednosti, ki pomeni izziv »tradicionalni« vednosti, kot to imenuje Drglin (2007, p. 111). Negativna doživljanja o institucionalni obravnavi po porodu je opisovalo šest intervjuvank, ki v ospredje postavljajo zlasti obsojajoči odnos nekaterih zdravstvenih delavcev. »A ja, vi ste pa tista, ki ste v hribih rodila, vi ne rabite tega, tako so mi večkrat rekli. Na trenutke se je čutilo, da nisva bila njihova.« (Hana, 32 let) »Zelo pogosto so me spraševali, kako to, da nisem prišla rodit v porodnišnico. To je bilo najbolj moteče, saj smo prve dni po porodu porodnice ne vem, kako naj rečem, bolj smo občutljive, bolj slabotne in nismo tako pri moči. […] Šele kasneje, ko sem se jaz toliko zbrala, da sem jim razložila, da smo bili tisti dan že na pregledu v porodnišnici in da so nas poslali nazaj domov, šele takrat so odnehali s temi vprašanji. To je bilo moteče, to ni bilo prijetno.« (Lucija, 37 let) Zaznavanje poroda med nosečnostjo: želje in pričakovanja Tovrstno ravnanje pa ima lahko trajne škodljive učinke na psihično, socialno in fizično počutje ženske (Davison, et al., 2015). i p j ( ) V okviru omejitev raziskave je treba omeniti, da so bile v raziskavo vključene porodnice, ki so nenačrtovano rodile zunaj porodnišnice med letoma 2004 in 2013. Dve od njih sta rodili leta 2004, zaradi česar bi lahko trdili, da je spomin na porod zbledel in da so podrobnosti pozabljene, a ugotovitve drugih avtorjev kažejo, da so spomini, vezani na porod, skozi leta konstantni (Sjöblom, et al., 2006), kar je najbrž tudi povezano z dejstvom, da se porodne izkušnje velikokrat podajo drugim v obliki pripovedi in se tako ohranjajo v njihovem spominu. Poleg tega je raziskava, opravljena na geografsko omejenem območju, v katerem je morda družbena zaznava nenačrtovanega poroda zunaj porodnišnice, ki na tem območju ni tako redek, drugačna v primerjavi z drugimi okolji, kar vpliva na posploševanje rezultatov. Med dejavniki, ki bi lahko vplivali na rezultate, je tudi vključenost prve avtorice, ki je sodelovala pri nekaterih porodih vključenih porodnic, zaradi česar se je poskušalo izpeljati nekatere ukrepe (receptivnost, neodvisna analiza besedila). Ugotovitve raziskave kažejo, da proces rojevanja zunaj porodnišnice zaznamujejo rituali, ki vključujejo pripravo pred odhodom od doma, porodne prakse, ki se morda razlikujejo od tistih v porodnišnici, in doživljanje rojevanja, na katerega pomembno vpliva stopnja zaupanja v zdravstveno osebje in prvi stik z novorojencem. Vzpostavljeno zaupanje v zdravstveno osebje in nenehna podpora sta veliko bolj krojila doživljanje poroda kot kraj rojevanja. S tem je povezana tudi avtonomija, ki so jo porodnice občutile, oziroma občutek nadzora nad dogajanjem. Lindgren in Erlandsson (2010) navajata, da prisotnost tega pomembno prispeva k celovitemu doživljanju poroda, kar pa ni nujno povezano s krajem rojevanja, temveč sposobnostjo porodnice, da nadzoruje svoje lastno vedenje in dejanja ljudi okrog sebe. Porodnice v raziskavi, ki so izkusile nenačrtovan porod zunaj porodnišnice ob prisotnem zdravstvenem osebju, izkušnjo poroda opisujejo kot pozitivno in opolnomočeno. Občutek nadzora nad dogajanjem in opolnomočenje se hitreje razvijeta, če imata porodnica in babica že pred porodom vzpostavljen pristen medsebojni odnos (Lindgren & Erlandsson, 2010; Hadjigeorgiou, et al., 2012), kot je bilo v večini primerov, ki so bili vključeni v raziskavo. Zaznavanje poroda med nosečnostjo: želje in pričakovanja Zaznavanje poroda med nosečnostjo: želje in pričakovanja Tako kot mnoge druge porodnice so se tudi pri intervjuvankah med nosečnostjo oblikovale želje in pričakovanja glede poroda. Sedem od njih neposredno pove, da del tega nikoli ni bila želja po načrtovanem porodu zunaj porodnišnice. Mateja (29 let) na primer pravi: j g Porodne zgodbe ostajajo trajen spomin. Tako sogovornice priznavajo, da se s spomini pogosto vračajo v čas poroda, ga v mislih podoživijo in vrednostno opredelijo. Tako slike poroda postanejo bolj jasne. Ugotovitve nekaterih raziskav (Coxon, et al., 2015; Erlandsson, et al., 2015) kažejo na to, da porodnice realno ocenijo nekatera svoja porodna pričakovanja, ki so jih razvile v nosečnosti šele pozneje, v poporodnem obdobju. V skladu s tem lahko sprejmemo tudi nekatere odločitve, ki so povezane z rojevanjem v prihodnje (Erlandsson, et al., 2015). Vse intervjuvanke v raziskavi so porod zunaj porodnišnice izkusile prvič in priznale, da se kljub upoštevanju časovne oddaljenosti do porodnišnice niso zavedale vseh morebitnih tveganj takšnega poroda. To potrjujejo tudi druge raziskave (Lee, et al., »V nosečnosti nikoli nisem razmišljala o tem, da ne bi rodila v porodnišnici.« V primerjavi z njimi dve priznavata, da sta razmišljali tudi o tem. Mojca (27 let) pripoveduje: V primerjavi z njimi dve priznavata, da sta razmišljali tudi o tem. Mojca (27 let) pripoveduje: »Zdaj, pred tem porodom, nisem razmišljala, da ne bi rodila v porodnišnici. V prvi nosečnosti, po pravici povedano, pa sem o tem razmišljala. Porod je nekaj naravnega, spontanega, le mi smo ga preveč zakomplicirali. Poglejte živali, pa krave, vse rodijo same.« g j p j Podobno kot Mojca (27 let) tudi Hana (32 let) poudarja: g j p j Podobno kot Mojca (27 let) tudi Hana (32 let) poudarja: »Namenoma nisem razmišljala, da ne bi rodila v porodnišnici. Sem pa razmišljala, kako bi vse skupaj potekalo, če bi se to res zgodilo. Zavestno pa se zato Kragelj, K. & Prosen, M., 2016. / Obzornik zdravstvene nege, 50(2), pp. 135–143. 141 2012), ki kažejo na to, da se zaznava dojemanja tveganj pri porodnici in zdravstvenem osebju (pri čemer so lahko razlike med različnimi profili, ki sodelujejo v obporodni oskrbi) zelo razlikuje, kar lahko vpliva na zagotavljanje zdravstvene oskrbe in celo izide poroda. njimi so zaradi tega čutile ponižanost in občutke krivde, kar ugotavljajo tudi v drugih raziskavah (Sjöblom, et al., 2012; Erlandsson, et al., 2015). Zaznavanje poroda med nosečnostjo: želje in pričakovanja To govori v prid kontinuirani babiški skrbi, ki se začne že v predporodnem obdobju z vzpostavitvijo partnerskega odnosa, ki temelji na sodelovanju in medsebojnem spoštovanju (Stanek Zidarič, et al., 2011) ter pozitivno vpliva na doživljanje poroda in na njegov izid (Sjöblom, et al., 2006). Ob nenačrtovanem porodu zunaj porodnišnice je kontinuirana babiška skrb, če jo izpostavimo v kontekstu raziskave, prej naključna kot namenska. Poleg znanja in odgovornosti, ki jo kontinuirana babiška skrb prinaša za izvajalce, namreč uresničevanje takšnega načina dela zahteva predvsem sistemske spremembe znotraj zdravstvenega varstva (Njenjić & Skela Savič, 2011; Stanek Zidarič, et al., 2011; Prelec et al 2014) Raziskovanje področja lahko v nadaljevanju še vedno vključuje kvalitativni metodološki pristop, ki bi proučeval izkušnje zdravstvenih delavcev, ki so prisotni ob nenačrtovanem porodu zunaj porodnišnice. Pomembno je tudi proučevanje organizacijskega vidika vodenja tovrstnih porodov oziroma organiziranja nujne medicinske pomoči, še posebej v tistih primerih, ko je življenje matere in/ ali ploda/novorojenca ogroženo in zdravstveno oskrbo krojijo časovna oddaljenost do najbližje porodnišnice, pomanjkljiva medicinsko-tehnična oprema (npr. inkubator) in drugi dejavniki. Zaključek Porod ni le biološki dogodek, temveč tudi edinstvena prelomnica v življenju ženske, zato je doživljanje porodne izkušnje mnogokrat določujoče. Mogoče še bolj intenzivno, če jo porod prehiti in je neizogiben, ne glede na kraj. Lahko je to sodobno opremljena porodna soba v porodnišnici, skromna urgentna ambulanta v zdravstvenem domu, daleč stran od porodnišnice, reševalno vozilo ali domača hiša. Ugotovitve nakazujejo, da v trenutku poroda in s tem njegovega doživljanja vstopajo v ospredje ljudje, ki so ob porodnici, in njihov odnos do nje. Čeprav se takrat morda res ne zavedajo vseh morebitnih tveganj, ki jih še dodatno povečujejo oddaljenost do najbližje porodnišnice in drugi dejavniki, se tega zavedajo zdravstveni delavci ob njej. Ne glede na vse vključene v raziskavo svojo porodno izkušnjo doživljajo kot pozitivno in zadovoljujočo. Ob tem se iz pripovedovanja intervjuvank zdi, da sta vzpostavljeno medsebojno zaupanje med porodnico in babico ter kontinuirana babiška skrb precej pomembnejša za ustvarjanje pozitivne porodne izkušnje kot kraj poroda, kar navajajo tudi Spaich in sodelavci (2013). Ne glede na to pa lahko sklepamo, da je zadovoljstvo s porodno izkušnjo vsaj posredno povezano tudi s krajem rojevanja, zlasti zaradi navajanja nekaterih intervjuvank o odsotnosti teh dveh faktorjev (medsebojno zaupanje in kontinuirana babiška skrb) v institucionalni (bolnišnični) obravnavi med nosečnostjo. 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https://openalex.org/W1893361383	https://europepmc.org/articles/pmc4745738?pdf=render	English	null	TRAIL-mediated killing of acute lymphoblastic leukemia by plasmacytoid dendritic cell-activated natural killer cells	Oncotarget	2,015	cc-by	9,750	TRAIL-mediated killing of acute lymphoblastic leukemia by plasmacytoid dendritic cell-activated natural killer cells Martin Lelaidier1,2, Yildian Dìaz-Rodriguez1,2, Martine Cordeau1,2, Paulo Cordeiro1, Elie Haddad1,2,3,4, Sabine Herblot1,3 and Michel Duval1,3,4 Bruneau, Centre de recherche du CHU Sainte-Justine, Montréal, Québec, Canada 2 Département de Microbiologie Infectiologie & Immunologie, Université de Montréal, Québec, Canada 3 Département de Pédiatrie, Université de Montréal, Québec, Canada 4 Département de Sciences Biomédicales, Université de Montréal, Québec, Canada Correspondence to: Sabine Herblot, email: sabine.herblot@umontreal.ca Keywords: pediatric acute lymphoblastic leukemia, natural killer cells, plasmacytoid dendritic cells, TRAIL, interferon-alpha Received: June 09, 2015 Accepted: July 12, 2015 Published: July 22, 2015 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. 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. 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. 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. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT), underscoring the need to improve the graft-versus- leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest, but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs, but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement, while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally, adoptive transfers of activated- pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL. Oncotarget, Vol. 6, No. 30 Oncotarget, Vol. 6, No. 30 www.impactjournals.com/oncotarget/ NK cell stimulation by TLR9-activated pDCs overcomes the resistance of ALL cells to NK cell killing NK cell stimulation by TLR9-activated pDCs overcomes the resistance of ALL cells to NK cell killing We tested whether NK cell stimulation by activated pDCs could enhance NK cell lytic functions against pre-B ALL. We assessed the susceptibility of three pre-B pediatric ALL cell lines to NK cell-mediated lysis, including KOPN8 cell line harboring the MLL translocation t(11;19). Human NK cells were isolated from adult volunteer’s peripheral blood samples, while pDCs were either freshly isolated from PBMC or in vitro differentiated from cord blood-CD34+ progenitors. Cytotoxic assays revealed that overnight stimulation of NK cells by pDCs significantly increased NK cell cytotoxic activity against all three pre-B ALL cell lines tested (Figure 1A). ALL specific lysis reached 60-80% at an E:T ratio of 5:1, depending on the target cell line. No significant differences were observed in NK cell activation depending on the pDC source (Supplemental Figure S1). Accordingly, we have previously showed that in vitro differentiated pDCs produce large amounts of IFN-α upon TLR stimulation and display the same phenotype as mature peripheral blood pDCs [26]. A direct TLR- 9 stimulation of NK cells by CpG ODN was ruled out, as CpG ODN alone did not increase NK cell cytotoxic activity against pre-B ALL cell lines (Supplemental Figure S2A). Moreover, unstimulated pDCs failed to enhance NK cell lytic activity, indicating that TLR-9 engagement on pDCs was required to enhance NK cell cytolytic functions (Supplemental Figure S2A). The lytic activity of TLR9- activated pDCs was also tested and, in the absence of NK cells, activated pDCs failed to induce pre-B ALL lysis, despite their high surface expression of TRAIL (Supplemental Figures S2B and C). While the role of NK cells in the GvL effect against acute myeloid leukemia (AML) has been well established, in vitro as well as clinical data showed that ALL blasts were more resistant to NK cell-mediated lysis. This is due not only to high levels of HLA class I expression, but also to low levels of stress-inducible proteins such as the ligands of the NKG2D receptor (MHC class I-related chains A and B – MICA/B and the members of the UL16- binding protein family), as well as low levels of adhesion molecules such as LFA-1 [16-18]. RESULTS Activating signals integrated by NK cells are counterbalanced by inhibiting signals conferred by the engagement of NK cell inhibitory receptors, such as killer immunoglobulin-like receptors (KIR) and the heterodimer NKG2A/CD94, through the recognition of various class I human leukocyte antigens (HLA class I) expressed by target cells [9, 12]. In order to induce NK cell-mediated lysis, inhibitory signals must be repressed (by HLA downregulation or KIR/HLA mismatch) or activating signals must be increased through NK cell activation by cytokines, dendritic cells and the engagement of activating receptors [9, 11]. The cytolytic pathways engaged by NK cells to kill target cells include the polarized release of cytotoxic granules and the induction of apoptosis via ligands of the tumor necrosis factor (TNF) family such as FAS-L and the TNF-related apoptosis-inducing ligand (TRAIL) [13-15]. TRAIL and FAS-L induce apoptosis through the cross-linking of their respective death receptors, DR4 (TRAIL-R1) DR5 (TRAIL-R2) and FAS, on target cells. NK cell stimulation by TLR9-activated pDCs overcomes the resistance of ALL cells to NK cell killing However, as recent studies provided evidence that activating signals can overcome NK cell inhibition by KIR ligands [19, 20], we explored new ways to activate NK cells in order to overcome ALL resistance to NK cell-mediated lysis. Plasmacytoid dendritic cells (pDCs) are an attractive therapeutic tool to increase the cytolytic activity of NK cells [21]. Upon stimulation of their Toll-like receptors (TLRs), pDCs produce high amounts of type I IFNs, as well as several cytokines and chemokines that act on NK cells to increase their lytic activity [22, 23]. Recent reports have provided evidence that pDCs initiate and coordinate specific anti-tumor responses for which NK cell cytotoxic activity is required [24, 25]. Moreover, their direct interactions with NK cells has been shown to trigger NK cell cytotoxic activity against NK cell-resistant malignancies [22]. INTRODUCTION Natural killer (NK) cells are the first lymphocytes to recover following allogeneic HSCT, when the residual leukemic burden is at its lowest [4-6]. As innate cytotoxic lymphocytes able to recognize and eliminate infected or tumor cells without prior sensitization, NK cells play a major role in the GvL effect post-HSCT [7]. Tumor cell killing by NK cells requires activating signals triggered by NK cell-activating receptors that recognize tumor- associated stress-induced molecules expressed by tumor cells [8-10]. NK cells express several activating receptors including the C-type lectin NKG2D, the DNAX accessory molecule-1 (DNAM-1) and natural cytotoxicity receptors (NCRs) such as NKp30, NKp44 and NKp46. NK cell Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite progress in chemotherapy and hematopoietic stem cell transplantation (HSCT), ALL remains a leading cause of death by cancer in children [1]. The cure rate of children with early relapse is only 50%, even after aggressive chemotherapy followed by allogeneic HSCT, highlighting the need for new therapeutic strategies [2, 3]. One of the most promising avenues of research is the increase of the graft-versus- leukemia (GvL) effect post-HSCT by harnessing the donor-derived immune system to eradicate leukemia. www.impactjournals.com/oncotarget Oncotarget 29440 involved in NK cell activation by TLR-9 activated pDCs as well as the cytolytic pathways involved in ALL lysis. involved in NK cell activation by TLR-9 activated pDCs as well as the cytolytic pathways involved in ALL lysis. cytotoxic activity can be further enhanced by cytokine stimulation, such as type I interferons (IFN) or by interactions with activated dendritic cells [11]. NK cell activation by TLR-9-activated pDCs is contact independent and IFN-α dependent Whether cell contact between activated pDCs and NK cells was required for enhancing NK cell lytic functions was then investigated. TLR-9 activated pDCs As IFN-γ production is another hallmark of NK cell activation, IFN-γ levels were measured following NK cell stimulation by activated pDCs. IFN-γ production was Figure 1: NK cell stimulation by TLR-9 activated pDCs overcomes ALL resistance to NK cell-mediated lysis and induces a unique activated phenotype. A. Purified human NK cells were stimulated overnight with IFN-α (1000 IU/mL), co-cultured with activated pDC (pDC+CpG) or unstimulated (NS). Cytotoxic assays were then performed against REH, KOPN8 and Nalm6 cells. Means of n experiments performed in triplicates (n = 5 for REH, n = 3 for Nalm6 and KOPN8) are shown. Means of specific lysis ± SEM are represented for each ALL cell line and indicated E:T ratio. Paired t-test was used for statistical comparison B.-C. Activated NK cell phenotype was assessed by flow cytometry and D. IFN-γ production by ELISA following overnight stimulation with IFN-α or co-culture with TLR9-activated pDCs. Box plots represent the distribution of MFI for CD69 and TRAIL immune-stainings or IFN-γ concentrations (first and third quartiles, median and standard error bars) (n = 9 independent experiments with different donors of NK cells and pDCs). Friedman’s test with Dunn’s post-test was used for statistical comparison. *p ≤ 0.001, p ≤ 0.01, p ≤ 0.05 Figure 1: NK cell stimulation by TLR-9 activated pDCs overcomes ALL resistance to NK cell-mediated lysis and induces a unique activated phenotype. A. Purified human NK cells were stimulated overnight with IFN-α (1000 IU/mL), co-cultured with activated pDC (pDC+CpG) or unstimulated (NS). Cytotoxic assays were then performed against REH, KOPN8 and Nalm6 cells. Means of n experiments performed in triplicates (n = 5 for REH, n = 3 for Nalm6 and KOPN8) are shown. Means of specific lysis ± SEM are represented for each ALL cell line and indicated E:T ratio. Paired t-test was used for statistical comparison B.-C. Activated NK cell phenotype was assessed by flow cytometry and D. IFN-γ production by ELISA following overnight stimulation with IFN-α or co-culture with TLR9-activated pDCs. Box plots represent the distribution of MFI for CD69 and TRAIL immune-stainings or IFN-γ concentrations (first and third quartiles, median and standard error bars) (n = 9 independent experiments with different donors of NK cells and pDCs). Friedman’s test with Dunn’s post-test was used for statistical comparison. High expression levels of TRAIL on NK cells stimulated by activated pDCs The phenotype of activated NK cells following overnight co-culture with activated pDCs was then assessed. A marked upregulation of the CD69 activation marker on pDC-stimulated NK cells (Figure 1B) was observed. The expression of TRAIL was also highly increased on NK cells following stimulation with activated pDCs (Figure 1C). In contrast, the expression of several other molecules remained unchanged, including FAS-L, the NK cell activating receptors DNAM-1, NKG2D, In this study, we used three pre-B ALL cell lines that differed in their levels of expression of NK cell activating ligands and HLA molecules. All of these cell lines were resistant to NK cell-mediated lysis in the absence of prior NK cell stimulation. We hypothesize that activation of NK cells by TLR-9 activated pDCs could overcome ALL resistance. We also explored the activating pathways www.impactjournals.com/oncotarget Oncotarget 29441 NKp30, NKp44 and NKp46 and the intracellular levels of perforin and granzyme B (Supplementary Figure S3). NK cell stimulation by activated pDCs thus led to the expression of membrane molecules on NK cells. significantly increased when NK cells were co-cultured with TLR-9-activated pDCs (Figure 1D), showing that activated pDCs induced not only NK cell cytolytic activity against ALL but also cytokine production. NK cell activation by TLR-9-activated pDCs is contact independent and IFN-α dependent p ≤ 0.001, p ≤ 0.01, p ≤ 0.05 by TLR-9 activated pDCs overcomes ALL resistance to NK cell-mediated lysis and i www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29442 igure 2: NK cell activation by TLR9-stimulated pDCs is contact independent and IFN-α dependent. A. Human NK were co-cultured overnight with activated pDCs in contact or separated with a semipermeable membrane (transwell, TW). No differ n NK cell cytolytic functions was observed in cytotoxic assays performed against REH cells at indicated E:T ratio. The percentag pecific lysis (means of 3 independent experiments ± SEM) is presented. B. The expression levels of CD69 and TRAIL were assesse ow cytometry. The MFI means ± SEM are presented (n = 9), paired t-test was used for statistical comparison p ≤ 0.05. C. Adding FN-α and anti-IFN-receptor antibodies during NK/pDCs co-culture inhibits the type I IFN pathway as assessed by intracellular stai f phosphorylated-STAT1 and STAT1. Histograms are shown with MFI for unstimulated NK cells (NS), isotype control and NK o-cultured with activated pDCs with or without specific blocking antibodies (n = 3) D CD69 and TRAIL upregulation was abolishe gure 2: NK cell activation by TLR9-stimulated pDCs is contact independent and IFN-α dependent. A. Human NK ere co-cultured overnight with activated pDCs in contact or separated with a semipermeable membrane (transwell, TW). No differ NK cell cytolytic functions was observed in cytotoxic assays performed against REH cells at indicated E:T ratio. The percenta ecific lysis (means of 3 independent experiments ± SEM) is presented B The expression levels of CD69 and TRAIL were assesse Figure 2: NK cell activation by TLR9-stimulated pDCs is contact independent and IFN-α dependent. A. Human NK cells were co-cultured overnight with activated pDCs in contact or separated with a semipermeable membrane (transwell, TW). No difference in NK cell cytolytic functions was observed in cytotoxic assays performed against REH cells at indicated E:T ratio. The percentage of specific lysis (means of 3 independent experiments ± SEM) is presented. B. The expression levels of CD69 and TRAIL were assessed by flow cytometry. The MFI means ± SEM are presented (n = 9), paired t-test was used for statistical comparison p ≤ 0.05. C. Adding anti- IFN-α and anti-IFN-receptor antibodies during NK/pDCs co-culture inhibits the type I IFN pathway as assessed by intracellular staining of phosphorylated-STAT1 and STAT1. Histograms are shown with MFI for unstimulated NK cells (NS), isotype control and NK cells co-cultured with activated pDCs with or without specific blocking antibodies (n = 3). D. www.impactjournals.com/oncotarget CD69 and TRAIL upregulation was abolished in presence of blocking anti-IFN-α antibodies. Histograms are shown with MFI (n = 3). E. Blockade of type I IFN pathway abrogates pDC- induced anti-leukemic activity of NK cells as assessed by cytotoxic assays (n = 4, E:T=5:1). Paired t-test was used for statistical comparison p ≤ 0.01. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29443 were co-cultured in contact with NK cells or separated by a semi-permeable porous membrane in transwell plates allowing for the diffusion of soluble factors. We observed that cytotoxic activity was similar for NK cells co-cultured with activated pDCs in contact or in transwell systems (Figure 2A). However, CD69 and TRAIL expression levels were lower on NK cells stimulated by activated pDCs in the absence of cell contact (Figure 2B). A combination of blocking monoclonal antibodies against IFN-α and the type I IFN receptor was then used. The blocking of the IFN-α pathway was first confirmed by intracellular staining of STAT1 and the phosphorylated form of STAT1 (Figure 2C). We observed that CD69 and TRAIL up-regulation on NK cells was entirely dependent on IFN-α production by activated pDCs (Figure 2D). The anti-leukemic activity of NK cells also was totally abrogated in the presence of blocking antibodies against the IFN-α pathway (Figure 2E). Although these results demonstrate that signaling through the type I IFN pathway is required to overcome pre-B ALL resistance to NK cell- mediated lysis, IFN-α stimulation alone was unable to reproduce the NK cell activation profile and NK cell lytic activity observed with co-culture with activated pDCs. Indeed, when used at a dosage corresponding to the amount of IFN-α produced by pDCs [27], IFN-α induced lower TRAIL and CD69 expression levels, lower IFN-γ production and lower lytic activity against ALL cells than with NK cells stimulated with activated pDCs (Figure 1). TLR9-activated pDCs enhance NK cell degranulation against ALL TLR9-activated pDCs enhance NK cell degranulation against ALL surface. To test whether inhibiting KIR/KIR-L interactions increases NK cell cytotoxicity, Nalm6 cells were chosen, since they expressed significant levels of HLA class I molecules, and we were able to find several matched NK cell donors (i.e. C1/C2 Bw6 genotype). However, mismatched donors could not be found due to the C1/ C2 genotype of this cell line. The addition of blocking antibodies against HLA class I molecules significantly increased the cytotoxic activity of pDC-activated NK cells as assessed by cytotoxic assays (Figure 3C). This result indicates that the enhancement of ALL killing by NK cells activated with pDCs can be further enhanced by a KIR- HLA mismatch between donor and recipient in the HSCT setting. NK cell degranulation was quantified by determining LAMP1 (Lysosomal-associated membrane protein-1)/CD107a surface expression on NK cells following effector:target contact.[28] We observed that unstimulated NK cells remained mainly negative for CD107a following incubation with ALL cells. However, the percentages of CD107a+ NK cells increased following NK cell activation. NK cells stimulated by activated pDCs exhibited a superior ability for degranulation than did IFN-α-stimulated NK cells (Figure 4A). Moreover, the proportion of CD107a+ NK cells was higher upon contact with REH and Nalm6 cells than with KOPN8 cells, indicating that factors present on target cells determine the capacity of NK cell degranulation. Figure 4: NK degranulation against ALL is increased following stimulation by TLR9-activated pDCs. A. Purified human NK cells were stimulated overnight with IFN-α (1000 IU/mL), co-cultured with activated pDC or unstimulated. NK cells were then incubated with REH, KOPN8 and Nalm6 target cells at ratio 1:1 for 4 h and CD107a expression was assessed by flow cytometry. Graphs represent means ± SEM of the percentages of CD107a positive cells among CD56+CD3- cells (n = 3). B. Unstimulated NK cells (NS) and pDC-stimulated NK cells were incubated with a blocking anti-NKG2D antibody or a control IgG1 before degranulation assay. NK cells were co-cultured with Nalm6 cells (ratio 1:1) for 4 h and the proportion of CD107a+ was assessed by flow cytometry. Percentages of CD107+ cells among CD3-CD56+ cells are presented with SEM (means of 4 independent experiments). Paired t-test was used for statistical comparison p ≤ 0.05, *p ≤ 0.01 Figure 4: NK degranulation against ALL is increased following stimulation by TLR9-activated pDCs. A. KIR/KIR-L blockade further enhances NK cell cytotoxicity against pre-B ALL As shown in Figures 3A and 3B, the three pre-B ALL cell lines used exhibit various genotypes of HLA class I and various levels of HLA class I molecules at their O t t 29444 i tj l / t t Figure 3: KIR/KIR-L blockade enhances NK cell cytotoxicity against pre-B ALL. A. Pre-B ALL cell lines express various levels of HLA class I molecules as assessed by flow cytometry. Representative histograms are shown. B. HLA class I genotype is shown for REH, KOPN8 and Nalm6 cells. C. Cytotoxic assays against Nalm6 (5:1 E:T ratio) were performed with KIR/HLA class I matched NK cells (Bw6 C1/C2 donors) in the presence or absence of HLA blocking antibodies. Means of 3 independent experiments performed in triplicates are shown. Means of specific lysis are represented with SEM. Paired t-test was used for statistical comparison p ≤ 0.05 Figure 3: KIR/KIR-L blockade enhances NK cell cytotoxicity against pre-B ALL. A. Pre-B ALL cell lines express various levels of HLA class I molecules as assessed by flow cytometry. Representative histograms are shown. B. HLA class I genotype is shown for REH, KOPN8 and Nalm6 cells. C. Cytotoxic assays against Nalm6 (5:1 E:T ratio) were performed with KIR/HLA class I matched NK cells (Bw6 C1/C2 donors) in the presence or absence of HLA blocking antibodies. Means of 3 independent experiments performed in triplicates are shown. Means of specific lysis are represented with SEM. Paired t-test was used for statistical comparison p ≤ 0.05 www.impactjournals.com/oncotarget Oncotarget 29444 TLR9-activated pDCs enhance NK cell degranulation against ALL Purified human NK cells were stimulated overnight with IFN-α (1000 IU/mL), co-cultured with activated pDC or unstimulated. NK cells were then incubated with REH, KOPN8 and Nalm6 target cells at ratio 1:1 for 4 h and CD107a expression was assessed by flow cytometry. Graphs represent means ± SEM of the percentages of CD107a positive cells among CD56+CD3- cells (n = 3). B. Unstimulated NK cells (NS) and pDC-stimulated NK cells were incubated with a blocking anti-NKG2D antibody or a control IgG1 before degranulation assay. NK cells were co-cultured with Nalm6 cells (ratio 1:1) for 4 h and the proportion of CD107a+ was assessed by flow cytometry. Percentages of CD107+ cells among CD3-CD56+ cells are presented with SEM (means of 4 independent experiments). Paired t-test was used for statistical comparison p ≤ 0.05, p ≤ 0.01 NKG2D, but not DNAM-1, plays a role in the killing of ligand-positive ALL cells i.e., Nectin-2 for all three cell lines and PVR for Nalm6 (Figure 5C), the addition of a blocking antibody against DNAM1 did not reduce the cytotoxic activity of activated NK cells against any of the three ALL cell lines (Figure 5D). The anti-DNAM1 antibody was, however, able to specifically inhibit NK cell cytotoxicity against other cancer cell lines (data not shown). Collectively, these results indicate that the NKG2D-mediated release of cytotoxic granules plays a role in NK cell lytic activity against some pre-B ALL cells, depending on the presence of the corresponding activating ligands. However, DNAM- 1 does not play any role even in the presence of its ligands on ALL cells. The NK cell activating receptors engaged by ALL cells to induce NK cell degranulation and cytolytic functions were then determined. We confirmed that pre-B ALLs expressed low levels of NKG2D ligands (MICA/B and ULBP-2), except for Nalm6 cells that expressed high levels of ULBP-2 (Figure 5A) [17, 29]. Accordingly, blocking NKG2D resulted in a decrease in Nalm6 specific lysis (Figure 5B) and a concomitant decrease in CD107+ expression on activated NK cells (Figure 4C). Although ALL cell lines expressed ligands for the DNAM1 receptor, Figure 5: NKG2D, but not DNAM1, plays a role in pre-B ALL lysis by activated NK cells. A. The expression of the NKG2D ligands, MICA/B and ULBP2 was assessed by flow cytometry. Histograms are shown for each pre-B ALL cell line (black line: isotype control; gray filled specific antibody). B. The addition of blocking anti-NKG2D antibody decreased NK cell cytolytic activity against Nalm6 but not against REH and KOPN8 cells (5:1 E:T ratio). Specific lysis means of 3 independent experiments performed in triplicates are represented ± SEM. Paired t-test was used for statistical comparison, p ≤ 0.01 C. The expression of the DNAM1 ligands, Nectin-2 and PVR, was assessed by flow cytometry (black line: isotype control; gray filled specific antibody). Representative histograms are shown for each pre-B ALL cell line. D. The addition of blocking anti-DNAM1 antibody did not affect NK cell cytolytic activity against all 3 pre-B ALL cell lines (5:1 E:T ratio). Specific lysis means of 3 independent experiments performed in triplicates ± SEM are represented. Figure 5: NKG2D, but not DNAM1, plays a role in pre-B ALL lysis by activated NK cells. A. The expression of the NKG2D ligands, MICA/B and ULBP2 was assessed by flow cytometry. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29445 NKG2D, but not DNAM-1, plays a role in the killing of ligand-positive ALL cells TRAIL-mediated apoptosis plays a major role in pre-B ALL killing by activated NK cells IFN, an important activator of NK cell lytic functions [33]. Besides the CD69 up-regulation and IFN-γ production already reported by others,[34, 35] we showed in this study that activated pDCs induce a strong upregulation of TRAIL on NK cells. Accordingly, we found that pre-B ALL cell lysis by pDC-activated NK cells is inhibited by the blockade of TRAIL/death receptor interactions. Given that a majority of pre-B ALL blasts express TRAIL-R1 and/or TRAIL-R2 [36] and that pre-B leukemia-initiating cells are sensitive to TRAIL-mediated apoptosis [37], our findings are of particular clinical interest to prevent ALL- relapse following HSCT. Our data, though, indicate that the release of cytotoxic granules by pDC-activated NK cells does not play a major role in pDC-induced NK cell cytotoxicity against ALL. The low expression of NK cell receptor ligands by ALL or the absence of co-activating signal required to triggered NK cell degranulation [38] may be responsible for the minor role of NK cell release of cytolytic granules. Future studies will delineate precisely the mechanisms of degranulation in the lysis of ALL cells by NK cells stimulated by activated pDCs. Since we observed that activated pDCs induced a high expression of TRAIL on activated NK cells, the role of TRAIL-mediated apoptosis in the killing of ALL by pDC-activated NK cells was investigated. We first observed that all three ALL cell lines expressed the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) (Figure 6A). We further showed that the specific lysis of pre-B ALL was inhibited in the presence of a blocking antibody against TRAIL. The lysis of KOPN8 was completely abrogated by TRAIL blockade, whereas lysis inhibition for REH and Nalm6 was 66% and 50 % respectively (Figure 6B). These results show that the NK cell-mediated cytotoxicity of some ALL cell lines, such as KOPN8, is completely dependent on TRAIL induced apoptosis, whereas TRAIL plays a major role in the killing of other cell lines, such as REH and Nalm6. NKG2D, but not DNAM-1, plays a role in the killing of ligand-positive ALL cells Histograms are shown for each pre-B ALL cell line (black line: isotype control; gray filled specific antibody). B. The addition of blocking anti-NKG2D antibody decreased NK cell cytolytic activity against Nalm6 but not against REH and KOPN8 cells (5:1 E:T ratio). Specific lysis means of 3 independent experiments performed in triplicates are represented ± SEM. Paired t-test was used for statistical comparison, p ≤ 0.01 C. The expression of the DNAM1 ligands, Nectin-2 and PVR, was assessed by flow cytometry (black line: isotype control; gray filled specific antibody). Representative histograms are shown for each pre-B ALL cell line. D. The addition of blocking anti-DNAM1 antibody did not affect NK cell cytolytic activity against all 3 pre-B ALL cell lines (5:1 E:T ratio). Specific lysis means of 3 independent experiments performed in triplicates ± SEM are represented. www.impactjournals.com/oncotarget Oncotarget 29446 Activated pDCs control human pre-B ALL in humanized mice Overcoming the resistance of pre-B ALL to NK cell-mediated lysis opens new therapeutic opportunities for post-transplant NK cell-based immunotherapy in children with high-risk ALL. Indeed, NK cells are the first lymphocytes to recover after HSCT during the first two months post-transplant, when leukemia burden is at its lowest after the radio-chemotherapeutic conditioning regimen [4-6]. Increasing the anti-leukemia function of donor-derived NK cells could therefore decrease the relapse rate and improve the survival of children with ALL. The production of IFN-γ by pDCs-activated NK cells is also expected to be beneficial in the post-HSCT context. Indeed, it has been shown in several models that IFN-γ enhances the GvL effect and also protects against GvHD [39]. Our in vitro and in vivo data pave the way for future post-transplant NK cell stimulation by adoptive transfer of third-party activated pDCs. Sufficient amounts of pDCs for adoptive transfer cannot be obtained from peripheral blood because adult blood contains very low numbers of pDCs [40]. Fortunately, we and others have described methods for in vitro expansion and differentiation of human pDCs from cord blood progenitors. Indeed, up to 107 functional human pDCs are obtained from cord blood units, making adoptive transfers of activated pDCs clinically feasible [26, 30, 41]. We and others recently described that human CD34+ precursors can be cultured and differentiated in pDCs in vitro [26, 30]. This method allows the production of high numbers of functional pDCs that can be activated by TLR ligands. We verified that injection of TLR-9- activated pDCs in humanized mice induced in vivo NK cell activation (Supplemental Figure 4). Humanized mice bearing human ALL were treated by repeated injections of TLR-9-activated pDCs. As shown by Kaplan-Meier survival curves, the leukemia onset was significantly delayed and 30% of the mice survived (Figure 7). These results indicate that activated pDCs are a unique therapeutic tool to activate NK cells and to control ALL in vivo. DISCUSSION We showed that the stimulation of NK cells by TLR- 9-activated pDCs overcomes the resistance of ALL cells to NK cell-mediated killing. We further demonstrated that high expression of TRAIL was a hallmark of pDC- activated NK cells and that TRAIL-induced apoptosis of target cells is the major cytolytic pathway involved in ALL lysis by pDC-activated NK cells. The inhibition of KIR engagement by HLA class-I molecules further enhanced ALL lysis by activated NK cells. Finally, we provide evidence that NK cell stimulation by activated pDCs can control ALL in vivo. In agreement with our results, TLR-9 agonist administration has been shown to induce anti-leukemia responses in mouse models [42, 43]. However, in the clinical HSCT setting, the therapeutic efficacy of TLR-9 ligands will depend on the presence of TLR-9 responsive pDCs. Studies have shown that pDC reconstitution is delayed following bone marrow transplantation, reaching normal blood pDC counts one year after transplantation [44, 45]. We recently reported that despite the rapid reconstitution of pDC counts after cord blood Cross-talk between NK cells and dendritic cells (DC) is responsible for enhanced activation of both cell types and increases their antitumor activity [22, 31, 32]. Among DC subsets, pDCs are the major producers of type I www.impactjournals.com/oncotarget Oncotarget 29447 6: TRAIL mediated apoptosis plays a major role in pre-B ALL lysis by activated NK cells. A. All 3 pre-B ALL ce press the death receptors, TRAIL-R1 and TRAIL-R2, as assessed by flow cytometry. Representative histograms are shown for eac LL cell line (black line: isotype control; gray filled specific antibody). B. NK cell-mediated cytotoxic activity against all 3 pre- ignificantly inhibited by the blockade of TRAIL/TRAIL-R interactions, using a specific blocking antibody against TRAIL (5:1 E aired t-test was used for statistical comparison, p ≤ 0.01, p ≤ 0.05 Figure 6: TRAIL mediated apoptosis plays a major role in pre-B ALL lysis by activated NK cells. A. All 3 pre-B ALL cell lines express the death receptors, TRAIL-R1 and TRAIL-R2, as assessed by flow cytometry. Representative histograms are shown for each pre-B ALL cell line (black line: isotype control; gray filled specific antibody). B. NK cell-mediated cytotoxic activity against all 3 pre-B ALL is significantly inhibited by the blockade of TRAIL/TRAIL-R interactions, using a specific blocking antibody against TRAIL (5:1 E:T ratio). DISCUSSION Paired t-test was used for statistical comparison, p ≤ 0.01, p ≤ 0.05 Figure 6: TRAIL mediated apoptosis plays a major role in pre-B ALL lysis by activated NK cells. A. All 3 pre-B ALL cell lines express the death receptors, TRAIL-R1 and TRAIL-R2, as assessed by flow cytometry. Representative histograms are shown for each pre-B ALL cell line (black line: isotype control; gray filled specific antibody). B. NK cell-mediated cytotoxic activity against all 3 pre-B ALL is significantly inhibited by the blockade of TRAIL/TRAIL-R interactions, using a specific blocking antibody against TRAIL (5:1 E:T ratio). Paired t-test was used for statistical comparison, *p ≤ 0.01, p ≤ 0.05 www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29448 or proliferation of regulatory T cells that decreases the GvL effect [46, 47]. IL-15 has recently been reported as a promising therapeutic cytokine to stimulate both innate and adaptive immunity against tumors [48]. However, IL- 15 also activates CD8+ cells, leading to a similar risk of GvHD. More importantly, a recent report has shown that IL-15 enhances pre-B ALL proliferation and dissemination in the central nervous system, precluding the use of this cytokine to increase NK cell functions in ALL patients [49]. Finally, our study shows that although the IFN-α pathway is absolutely required for pDC-induced NK cell activation, IFN-α alone is not able to reproduce TRAIL upregulation on NK cells and NK cell lytic activity against ALL observed with pDC-activated NK cells. This result suggests that other soluble factors participate in NK cell transplantation, these pDCs display impaired IFN-α production in response to TLR-9 stimulation [26]. A low number or impaired function of pDCs during the first months after HSCT is thus a limitation for the use of TLR-9 agonists in this setting. Fortunately, our in vivo data show the feasibility of adoptive transfer of activated pDCs and their efficacy in controlling leukemia. Further pre- clinical experiments are currently underway to increase the efficacy of this approach. pDCs have advantages over other candidates for NK cell activation after HSCT such as cytokine stimulation with IL-2, IL-15 or IFN-α. The systemic administration of inflammatory cytokines such as IL-2 induces severe side effects related to T lymphocyte proliferation and activation, i.e., severe graft-versus-host disease (GvHD) Figure 7: Activated pDC infusions delay leukemia onset in humanized mice. A. Experimental design. Irradiated newborn NSG mice were transplanted with 104 human cord blood-derived CD34+ cells. 105 REH ALL cells were intravenously injected in humanized mice 5-6 weeks after transplantation, followed by four weekly injections of activated pDCs or saline solution for control mice (CTL). B. Survival curves of ALL-bearing humanized mice treated with activated pDCs or saline solution injections. Mice were sacrificed after overt leukemia onset. Flow cytometry analysis of bone marrow samples confirmed complete leukemia involvement. Log-rank test was used to compare survival. Figure 7: Activated pDC infusions delay leukemia onset in humanized mice. A. Experimental design. Irradiated newborn NSG mice were transplanted with 104 human cord blood-derived CD34+ cells. www.impactjournals.com/oncotarget 105 REH ALL cells were intravenously injected in humanized mice 5-6 weeks after transplantation, followed by four weekly injections of activated pDCs or saline solution for control mice (CTL). B. Survival curves of ALL-bearing humanized mice treated with activated pDCs or saline solution injections. Mice were sacrificed after overt leukemia onset. Flow cytometry analysis of bone marrow samples confirmed complete leukemia involvement. Log-rank test was used to compare survival. Figure 7: Activated pDC infusions delay leukemia onset in humanized mice. A. Experimental design. Irradiated newborn NSG mice were transplanted with 104 human cord blood-derived CD34+ cells. 105 REH ALL cells were intravenously injected in humanized mice 5-6 weeks after transplantation, followed by four weekly injections of activated pDCs or saline solution for control mice (CTL). B. Survival curves of ALL-bearing humanized mice treated with activated pDCs or saline solution injections. Mice were sacrificed after overt leukemia onset. Flow cytometry analysis of bone marrow samples confirmed complete leukemia involvement. Log-rank test was used to compare survival. NK cell stimulation Three pediatric B-cell precursor ALL (pre-B ALL) cell lines resistant to NK cell-mediated lysis were used in this study: REH t(12;21), Nalm6 t(5;12) and KOPN- 8 t(11;19) that harbors a rearrangement of the MLL gene (DSMZ Braunschweig, Germany). Cell lines were maintained in RPMI-1640 (Wisent, Saint-Bruno, QC) medium supplemented with 10% heat-inactivated FBS (Wisent, Canada) and 1% Antibiotic-Antimycotic (Life Technologies, Burlington, ON) at 37°C in a 5% CO2 atmosphere. All cell lines were negative for mycoplasma contamination, as assessed by PCR using the Universal Mycoplasma Detection Kit (ATCC, Manassas, VA). NK cells were plated in a 96-well round-bottom plate (2×106 cells/mL) and incubated overnight without stimulation or with human IFN-α (1000 UI/ml) or co- cultured with pDCs (Lin-HLA-DR+CD123+) (ratio NK:pDC of 10:1) and CpG-A ODN2216 (10μg/ml) (Invivogen, San Diego, CA) at 37°C in a 5% CO2 atmosphere. Of note, pDCs and NK cells were not prepared from the same donor. In some experiments, 24-well microplates equipped with a transwell insert (0.4μm, Greiner bio-one) were used to prevent direct contact between NK cells and pDCs. For IFN-α signaling neutralization assays, NK cells were incubated for 30 minutes with neutralizing anti-IFN-α/β receptor Chain2 and anti-IFN-α antibodies (20μg/ml each) (MMHAR-2 and MMHA-2 respectively; PBL Assay Science, Piscataway, NJ), prior to the addition of IFN-α or pDCs and overnight incubation. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29449 Uppsala, Sweden). CD34+ progenitors were isolated by positive selection using CD34 antibody-conjugated magnetic microbeads and MiniMACS columns (Miltenyi Biotec, San Diego, CA) and were seeded at a density of 2 x 105 cells/mL in serum-free expansion medium (StemSpanTM SFEM, StemCell Technologies, Vancouver, BC). The cells were expanded for 7 days with recombinant human stem cell factor (10 ng/mL), thrombopoietin (50 ng/mL) and FLT3-ligand (100 ng/mL) (all from R&D System, Minneapolis, MN or Miltenyi Biotec). Expanded CD34+ hematopoietic stem cells (HSCs) were then cultured with rhIL-7 (10ng/mL, Cytheris, France), TPO and Flt3-ligand for 7 days of differentiation. The cultures were refreshed every 2 days and maintained for a total of 14 days at 37°C in a humidified incubator with 5% CO2. Cells were stained with the labelled mouse anti-human antibodies and pDCs (Lin-HLA-DR+CD123+) were sorted by flow cytometry (FACS Aria, BD Biosciences, San Jose, CA). The functional characteristics of expanded pDCs have been described elsewhere [26]. activation induced by activated pDCs. The characterization of these cytokines and chemokines is underway and will be of particular importance for future clinical applications. p p pp Although several laboratory and clinical studies have shown that KIR mismatch alone is not sufficient to activate NK cells and eradicate ALL [50-54], we showed here that blockade of the HLA-KIR recognition further increases ALL lysis by pDC-activated NK cells. This finding has important clinical implications. NK cell stimulation by activated pDCs can be expected to be more efficient in the context of a KIR mismatch between donor and recipient. Similarly, it has recently been reported that the cytotoxicity of expanded NK cells against pediatric pre-B ALL was higher when donor NK cells were KIR mismatched with their targets [55]. Together, these results indicate that post-HSCT NK cell-based immunotherapy has a greater chance of success if donor derived NK cells are not inhibited by KIR/HLA interactions. In conclusion, our data show that the resistance of ALL to NK cell lysis can be overcome by the use of activated pDCs to increase NK cell lytic functions. These data open new opportunities of post-transplant immunotherapy based on NK cell stimulation by activated pDCs with many advantages over other methods of post- transplant NK cell stimulation. Pre-clinical experiments are currently underway to refine this approach. NK cell and pDC isolation from peripheral blood Peripheral blood mononuclear cells (PBMC) from healthy volunteers were isolated by Ficoll-PaquePLUS density gradient centrifugation. NK cells and pDCs were then isolated by negative selection with the EasySep® NK Cell Enrichment Kit and the EasySep® pDC Enrichment Kit (StemCell Technologies), respectively. The purity of NK cells and pDCs was always above 95%. pDC expansion and differentiation from cord blood CD34+ cells Human pDCs were expanded and differentiated from purified cord blood-CD34+ progenitors as described [26]. Briefly, Cord blood units were obtained from the CHU Sainte-Justine Research Center cord blood bank with approval from the Institutional Review Board. Mononuclear cells were isolated by gradient centrifugation on Ficoll-PaquePLUS (GE Healthcare Bio-Science AB, Degranulation assays CD107a staining was used to assess NK cell degranulation against ALL cell lines as previously described.[28] Briefly, after an overnight incubation with activated pDCs or IFN-α, NK cells were harvested and cultured with ALL cells at 1:1 E:T ratio, in the presence of PE-conjugated CD107a- specific mAb (BD Biosciences). Monensin (Golgi-stop, BD Biosciences) was added to the co-culture one hour after the beginning of the incubation to avoid the degradation of reinternalized proteins from the surface. Cells were incubated for three additional hours and then stained with APC-conjugated anti-CD56 and PE-Cy7-conjugated anti-CD3 before being analyzed by flow cytometry. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29450 Dosage of IFN-γ production The ability of NK cells to kill pre-B ALL cell lines was assessed by cytotoxic assays using flow cytometry with the LIVE/DEAD® Cell-mediated cytotoxicity kit (Life Technologies, Invitrogen). Target cells were labelled with the green-fluorescent membrane dye DiOC18. NK cells were harvested after overnight stimulation. NK and target cells were then plated in triplicates at different Effector:Target (E:T) ratios (ranging from 1:5 to 5:1) in a 96-well coned plate. Cells were briefly spun to provide immediate contact between NK cells and targets and then incubated for two hours at 37°C. Dead cells were then stained with propidium iodide (PI) and counting beads were added. Flow cytometry analysis was performed using an LSR Fortessa cytometer (BD Bioscience, San Jose, CA) and data were analyzed with FlowJo software (Tree Star, Ashland, OR). Absolute numbers of live target cells were assessed (DiOC+PI-) and data were expressed as the percentage of specific lysis calculated by the following formula: specific lysis (%) = [(#absolute live cells – #experimental live cells)/(#absolute live cells)] × 100. For blocking experiments, after Fc receptor saturation with purified mouse IgG1, NK cells were incubated with 30μg/ml of purified NA/LE mouse anti-human CD253 (TRAIL) mAb or purified NA/LE mouse anti-human CD226 (DNAM-1) mAb (BD Pharmingen, San Jose, CA) or anti-human NKG2D (Amgen, Mississauga, ON) for 30 minutes at RT as previously described.[56] NK cells were stimulated overnight as described above. Supernatants were collected and stored at -80°C. IFN-γ dosage was performed by enzyme-linked immunosorbent assay (ELISA) (PBL InterferonSource, Piscataway, NJ). In vivo control of leukemia Nod/Scid/IL-2Rγ-/- mice were purchased from the Jackson Laboratory and maintained in pathogen-free conditions. Humanized mice were generated as previously described [57]. Briefly, newborn (1-3-day-old) mice received sub-lethal (1 Gy) total body irradiation from an X-ray source (Faxitron CP160), and were injected intra- hepatically with 104 human CD34+ purified from cord blood units. Protocols for generating humanized mice were approved by our local Animal Care Committee according to the guidelines of the Canadian Council on Animal Care in Science. To promote human NK cell differentiation, humanized mice received human IL-15/IL-15Rα-Fc complex (ALT803, Altor Biosciences [58]) once a week for seven weeks starting six weeks after transplantation. To reproduce human ALL in humanized mice, 104 REH cells were injected intravenously 8 weeks after transplantation, followed 24 h later by infusions of activated pDCs (105 pDCs per mouse). pDC injections were repeated once a week for five weeks. A group of humanized mice injected with REH cells and treated with saline injection was used as control. Leukemia development was monitored by weekly bleeding and mice were sacrificed when overt leukemia signs were reached. Immunophenotypic analysis by flow cytometry NK cell phenotype was assessed by flow cytometry after overnight stimulation with IFN-α or TLR9-activated pDCs. Unstimulated NK cells were used as controls. All conjugated monoclonal antibodies were purchased from BD Biosciences or Biolegend. We used FITC-conjugated anti-CD69 and anti-Granzyme B; PE-conjugated anti- NKp44, anti-Fas-ligand, anti-perforin, anti-NKG2D, anti- DNAM-1 and anti-TRAIL; alexa-fluor® 647-conjugated anti-NKp30; brilliant violet 421™-conjugated anti- NKp46. Granzyme B and perforin protein expression were assessed by intracellular staining as previously described. [28] The expression of death receptors, NKG2D and DNAM-1 ligands as well as HLA class I molecules was assessed on pre-B ALL cell lines using APC-conjugated anti-DR4, PE-conjugated anti-DR5, PE-conjugated anti-PVR, PE-conjugated anti-Nectin2, PE-conjugated anti-MICA/MICB, PE-conjugated anti-ULBP2 and PE- conjugated anti-HLA class I. Flow cytometry analysis was performed with LSR Fortessa cytometer and data were analyzed with FlowJo software. GRANT SUPPORTS 9. Bakker AB, Wu J, Phillips JH and Lanier LL. NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals. Human immunology. 2000; 61:18-27. This research study was conducted with support from C17 and funded by the Childhood Cancer Canada Foundation, Charles-Bruneau Foundation and Canadian Institutes of Health Research (CIHR). MC and YRD were supported by the Fondation du CHU Sainte-Justine/ Fondation des Étoiles. YRD was also supported by the Fonds de la Recherche du Québec - Santé (FRQS). 10. Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R and Moretta L. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annual review of immunology. 2001; 19:197-223. 11. Vivier E and Ugolini S. Natural killer cells: from basic research to treatments. Frontiers in Immunology. 2011; 2:1- 4. ACKNOWLEDGMENTS 5. Renard C, Barlogis V, Mialou V, Galambrun C, Bernoux D, Goutagny MP, Glasman L, Loundou AD, Poitevin-Later F, Dignat-George F, Dubois V, Picard C, Chabannon C, Bertrand Y and Michel G. Lymphocyte subset reconstitution after unrelated cord blood or bone marrow transplantation in children. British journal of haematology. 2011; 152:322- 330. The authors would like to express their gratitude to Dr. Françoise Le Deist for her critical reading of this manuscript and helpful comments and Lubomir Alexandrov for sharing his expertise in statistical analysis. They wish to thank Amgen Inc. for generously providing anti-NKG2D blocking antibody, Cytheris, France, for supplying human recombinant IL-7 and Dr. Wong from Altor Bioscience for supplying with human IL-15/IL- 15Rα-Fc complex. They would also like to thank Ines Boufaied and Danièle Gagné for cell sorting assistance and the clinical research team at the Centre de Cancérologie Charles-Bruneau. 6. Foley B, Felices M, Cichocki F, Cooley S, Verneris MR and Miller JS. The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT). Immunological reviews. 2014; 258:45-63. 7. Moretta A, Bottino C, Mingari MC, Biassoni R and Moretta L. What is a natural killer cell? Nature immunology. 2002; 3:6-8. Statistics All in vitro experiments were repeated at least three times with different blood donors for NK cell and pDC www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 29451 2. Pui CH, Gaynon PS, Boyett JM, Chessells JM, Baruchel A, Kamps W, Silverman LB, Biondi A, Harms DO, Vilmer E, Schrappe M and Camitta B. Outcome of treatment in childhood acute lymphoblastic leukaemia with rearrangements of the 11q23 chromosomal region. Lancet. 2002; 359:1909-1915. purification and all results are presented as the mean of n experiments to eliminate donor-derived variation. Animal experiments were performed three times with five mice in each group; the survival curve presented includes all the 15 mice in treated and untreated groups. Animals were allocated into groups after NK cell numbering to ensure comparable NK cell reconstitution among groups. Animal experiments were not blinded. 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https://openalex.org/W4310480134	https://www.frontiersin.org/articles/10.3389/fpsyt.2022.978763/pdf	English	null	Exploring changes in depression and radiology-related publications research focus: A bibliometrics and content analysis based on natural language processing	Frontiers in psychiatry	2,022	cc-by	9,493	Exploring changes in depression and radiology-related publications research focus: A bibliometrics and content analysis based on natural language processing OPEN ACCESS EDITED BY Jiliang Fang, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, China REVIEWED BY Raman Kumar, Guru Nanak Dev Engineering College, India Dan Ma, Xiyuan Hospital, China Academy of Chinese Medical Sciences, China CORRESPONDENCE Lingyu Kong kong_lingyu@csu.edu.cn SPECIALTY SECTION This article was submitted to Computational Psychiatry, a section of the journal Frontiers in Psychiatry RECEIVED 26 July 2022 ACCEPTED 14 November 2022 PUBLISHED 30 November 2022 Kangtao Wang1,2, Fengbo Tan1,2, Zhiming Zhu3 and Lingyu Kong3 Kangtao Wang1,2, Fengbo Tan1,2, Zhiming Zhu3 and Lingyu Kong3* 1Department of General Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, China, 2National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China, 3Department of Radiology, Xiangya Hospital, Central South University, Changsha, Hunan, China Objective: This study aims to construct and use natural language processing and other methods to analyze major depressive disorder (MDD) and radiology studies’ publications in the PubMed database to understand the historical growth, current state, and potential expansion trend. CITATION Wang K, Tan F, Zhu Z and Kong L (2022) Exploring changes in depression and radiology-related publications research focus: A bibliometrics and content analysis based on natural language processing. Methods: All MDD radiology studies publications from January 2002 to January 2022 were downloaded from PubMed using R, a statistical computing language. R and the interpretive general-purpose programming language Python were used to extract publication dates, geographic information, and abstracts from each publication’s metadata for bibliometric analysis. The generative statistical algorithm “Latent Dirichlet allocation” (LDA) was applied to identify speciﬁc research focus and trends. The unsupervised Leuven algorithm was used to build a network to identify relationships between research focus. Front. Psychiatry 13:978763. doi: 10.3389/fpsyt.2022.978763 © 2022 Wang, Tan, Zhu and Kong. 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. Results: A total of 5,566 publications on MDD and radiology research were identiﬁed, and there is a rapid upward trend. TYPE Original Research PUBLISHED 30 November 2022 DOI 10.3389/fpsyt.2022.978763 TYPE Original Research PUBLISHED 30 November 2022 DOI 10.3389/fpsyt.2022.978763 TYPE Original Research PUBLISHED 30 November 2022 DOI 10.3389/fpsyt.2022.978763 Exploring changes in depression and radiology-related publications research focus: A bibliometrics and content analysis based on natural language processing The top-cited publications were 11,042, and the highly-cited publications focused on improving diagnostic performance and establishing imaging standards.Publications came from 76 countries, with the most from research institutions in the United States and China. Hospitals and radiology departments take the lead in research and have an advantage. The extensive ﬁeld of study contains 12,058 Medical Subject Heading (MeSH) terms. Based on the LDA algorithm, three areas were identiﬁed that have become the focus of research in recent years, “Symptoms and treatment,” “Brain structure and imaging,” and “Comorbidities research.” Conclusion: Latent Dirichlet allocation analysis methods can be well used to analyze many texts and discover recent research trends and focus. In Frontiers in Psychiatry 01 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 10.3389/fpsyt.2022.978763 the past 20 years, the research on MDD and radiology has focused on exploring MDD mechanisms, establishing standards, and constructing imaging methods. Recent research focuses are “Symptoms and sleep,” “Brain structure study,” and “functional connectivity.” New progress may be made in studies on MDD complications and the combination of brain structure and metabolism. major depressive disorder, radiology, bibliometric analysis, Latent Dirichlet allocation, machine learning Research design Current research shows that MDD is closely related to brain structure and function changes. With the development of radiology, signiﬁcant progress has been made in exploring the neurobiological mechanisms of MDD (7). Radiology can not only use a variety of non-invasive methods to detect and analyze brain structure but also explore MDD brain function and brain connectivity through functional magnetic resonance imaging (fMRI) (8). In addition, techniques such as positron emission tomography (PET) and magnetic resonance spectroscopy (MRS) can track a variety of substances, thereby revealing the relationship between depression and brain metabolism (9, 10). Therefore, radiology has an essential function as a diagnostic and research tool to explore the pathogenesis of MDD. The study design was based on compliance with the fundamental principles of bibliometrics (14, 15). The study employed a two-stage structured approach to bibliometric analysis and visual assessment of published scientiﬁc literature. The PubMed database is a biomedical specialty database containing compound search strategies and is a free, publicly available database. The PubMed database is used for research purposes, and PubMed contains Application Programming Interface (API) that can export abstracts. In addition, all MDD and its radiology-related publications must be searched as thoroughly as possible for research purposes. The text volume of MDD and Radiology publications worldwide has exceeded tens of millions of words, and it is impossible to extract the information from the papers by reading. The correlation between papers, continuity, and other complex internal correlation information has exceeded the scope of human subjective understanding. Natural language processing (NLP) is a machine learning technology tool specializing in text processing (11). It has been widely used in medical research. It can analyze many texts, extract research topics, and visually facilitate researchers to understand massive texts and extract internal correlations (12, 13). Frontiers in Psychiatry Introduction various algorithms, such as Latent Dirichlet allocation (LDA), to discover changes in research focus and future predictions. This research aims to use the NLP method and multiple algorithms, such as LDA, to conduct a bibliometric analysis of all publications related to depression and radiology in the past 20 years. This study hopes to extract relevant research focus, discover changes in research content, predict possible future breakthroughs direction, and summarize the research of the last 20 years. Major depressive disorder (MDD) is an aﬀective psychosis characterized by depression with symptoms such as sleep disturbance, anxiety, and physical discomfort (1, 2). MDD is one of the leading causes of disability and suicide, with more than 800,000 suicides worldwide (3). It is the fourth leading disabling disease globally (4). To make matters worse, the pathogenesis of MDD is not well understood, and treatment options are limited (5). Furthermore, under the inﬂuence of the COVID- 19 pandemic, the number of MDD patients and the degree of deterioration will increase (6). MDD will cause great trouble to human health in the foreseeable future. The number of publications of radiology and major depressive disorder-related research is increasing every year From 2002 to 2022, 5,566 relevant publications were included, as shown in the ﬂowchart (Figure 1A). A total of 237 publications were manually excluded, and the exclusion criteria were missing data if the publications were conference abstracts, conference papers, corrections, book reviews, or news items. The number of remaining 5,566 publications was further reduced due to the manual exclusion of non-English publications or publications with incomplete abstracts, resulting in a remaining 5,409 publications. The LDA bibliometric modeling approach further analyzed these selected publications. Results The R language1 (version: 4.1.3) was used to access the PubMed database through the pubquery package and download publications limited to publication dates from January 1, 2002, to January 1, 2022. In order to include the total number of possible publications, the most comprehensive search strategies “depression” and “radiology” were adopted and the database was eventually downloaded. For speciﬁc search terms, please refer to the Supplementary material. Tables and visualizations are based on R and Excel (Microsoft Corporation, Redmond, WA, USA). Search and download code is available in R through the easyPubMed package.2 Inclusive and exclusive criteria The LDA-related code is available in the Supplementary material (see footnote 2). TABLE 1 Major depressive disorder (MDD) and radiology publications assortment steps. TABLE 1 Major depressive disorder (MDD) and radiology publications assortment steps. including publication year, geographic information, abstract, study type, and Medical Subject Heading (MeSH) terms. MeSH terms are a controlled vocabulary dictionary produced by the National Library of Medicine.4 The PubMed database uses MeSH terms to summarize the topic content of each publication, and MeSH terms were used to conduct a preliminary analysis of the development of research topics. LDA is an unsupervised topic model algorithm built according to previous research methods (16). LDA identiﬁes abstracts for all publications and sets the number of identiﬁed topics to 50. Criteria for topic number selection are based on appropriate perplexity, redundancy, and legibility levels. LDA is used to identify more speciﬁc research topics based on algorithmically computed topic probabilities and the opinions of all authors, ultimately manually determining the topic of each article. Heatmaps were generated in R to describe research topics and publication dates. Cluster analysis was performed using the Louvain algorithm in Gephi software5 (version 0.9.4) to build topic networks and determine relationships between topics. The two topics with the highest attribution probability in each article were used to calculate the number of co-occurrences in each document (17). The LDA-related code is available in the Supplementary material (see footnote 2). Exploration steps Query on pubMed Description obtain changes in the focus of research topics in publications in this ﬁeld and their correlations. Inclusive and exclusive criteria Table 1 shows the steps to obtain the complete raw data in the PubMed database. All MDD Radiology-related publications were published between January 1, 2002, and January 1, 2022, resulting in 5,803 publications. Missing data, conference abstracts, conference proceedings, book reviews, and news items were excluded, and ultimately 5,566 publications were included in the bibliometric analysis (Figure 1). Details of inclusion and exclusion are shown in Table 2. After excluding publications with the most non-English and incomplete abstracts, the ﬁnal 5,406 publications were analyzed by the LDA algorithm to This study aimed to perform a bibliometric analysis of all publications related to depression and radiology over the past 20 years. Further build and use NLP methods and 02 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 10.3389/fpsyt.2022.978763 TABLE 1 Major depressive disorder (MDD) and radiology publications assortment steps. Exploration steps Query on pubMed Description 1 Depression "depressed"[All Fields] OR "depression"[MeSH Terms] OR "depression"[All Fields] OR "depressions"[All Fields] OR "depression’s"[All Fields] OR "depressive disorder"[MeSH Terms] OR ("depressive"[All Fields] AND "disorder"[All Fields]) OR "depressive disorder"[All Fields] OR "depressivity"[All Fields] OR "depressive"[All Fields] OR "depressively"[All Fields] OR "depressiveness"[All Fields] OR "depressives"[All Fields] 2 AND Stop word: and 3 Radiology "radiology"[MeSH Terms] OR "radiology"[All Fields] OR "radiography"[MeSH Terms] OR "radiography"[All Fields] OR "radiology’s"[All Fields] 4 Data limiation (2002:2022[pdat]) including publication year, geographic information, abstract, study type, and Medical Subject Heading (MeSH) terms. MeSH terms are a controlled vocabulary dictionary produced by the National Library of Medicine.4 The PubMed database uses MeSH terms to summarize the topic content of each publication, and MeSH terms were used to conduct a preliminary analysis of the development of research topics. LDA is an unsupervised topic model algorithm built according to previous research methods (16). LDA identiﬁes abstracts for all publications and sets the number of identiﬁed topics to 50. Criteria for topic number selection are based on appropriate perplexity, redundancy, and legibility levels. LDA is used to identify more speciﬁc research topics based on algorithmically computed topic probabilities and the opinions of all authors, ultimately manually determining the topic of each article. Heatmaps were generated in R to describe research topics and publication dates. Cluster analysis was performed using the Louvain algorithm in Gephi software5 (version 0.9.4) to build topic networks and determine relationships between topics. The two topics with the highest attribution probability in each article were used to calculate the number of co-occurrences in each document (17). 4 https://www.ncbi.nlm.nih.gov/mesh 5 https://gephi.org/ 1 https://www.r-project.org/ 2 https://cran.r-project.org/web/packages/easyPubMed/index.html 3 https://www.python.org/ 3 https://www.python.org/ Latent Dirichlet allocation and algorithms and analytical methods After the publication acquisition, the publication was processed and analyzed based on the ﬂowchart in Figure 1A. Guido van Rossum created Python in the Netherlands in the early 1990s at Stichting Mathematisch Centrum. Python provides eﬃcient data structures and simple and eﬀective object- oriented programming and is widely used in machine learning and NLP. Python3 (version 3.10.4) extracted publication data, Eighty-nine publications related to depression and radiology were found in 2002, which increased to 714 publications in 2021 (Figure 1B). An average of 278 publications were published annually, representing an average annual growth rate of 14.4%. Using exponential function ﬁtting found that R2 = 0.96, and the composite function y = 69.748e0.1101x can describe the number Frontiers in Psychiatry Frontiers in Psychiatry 03 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 FIGURE 1 The number of publications on radiology and depression has increased rapidly over the past 20 years. (A) Using the search terms depression and radiology in the PubMed database, download publications through the R pubquery package. Missing data or when the publication was a meeting abstract, proceedings paper, a correction, a book review, or a news item were manually excluded, and ﬁnally, 5,566 publications were included in the general analysis. LDA analyzed 5,409 publications. (B) Publications analyzed by LDA, Python. Data were visualized using Excel. The number of publications is shown each year, and y = 69.748e0.1101x is the ﬁtted function. FIGURE 1 The number of publications on radiology and depression has increased rapidly over the past 20 years. (A) Using the search terms depression and radiology in the PubMed database, download publications through the R pubquery package. Missing data or when the publication was a meeting abstract, proceedings paper, a correction, a book review, or a news item were manually excluded, and ﬁnally, 5,566 publications were included in the general analysis. LDA analyzed 5,409 publications. (B) Publications analyzed by LDA, Python. Data were visualized using Excel. The number of publications is shown each year, and y = 69.748e0.1101x is the ﬁtted function. of publications well. It is expected that there will be 750 and 840 publications in 2022 and 2023. to 110 in 2017–2021, and the Multicenter Study publications also increased considerably, indicating that a large number of clinical trials in this ﬁeld have been published. Based on the data, it can be assumed that more than 130 clinical trials are in progress. Latent Dirichlet allocation and algorithms and analytical methods In order to further explore the research types, eight categories based on the classiﬁcation of publications provided by the PubMed database were identiﬁed: Case Reports, Review, Comparative Study, Randomized Controlled Trial, Multicenter Study, Clinical Trial and Study, Meta-Analysis, Systematic Review. In the past 20 years, the number of all types of research has increased signiﬁcantly, the most notable is Review, from 47 in 2002–2006 to 182 in 2017–2021(Supplementary Table 1 and Figure 2). On the other hand, the number of Randomized Controlled Trial published from 19 in 2002–2006 High-cited publications focus on establishing criteria for the diagnosis of major depressive disorder and constructing imaging methods High-cited publications are considered critical publications for research in related ﬁelds. Internal citations were also given more importance among the publications included in the analysis. It was found that the total number of citations exceeded 60,000, of which 105 were internally cited the most, accounting for 2% of the total included publications (Table 4 and Supplementary Table 2). The most internally cited is N Tzourio-Mazoyer’s macroscopic brain anatomy by MRI, totaling 11,042 citations (28). The results suggest that the most cited publication can be roughly divided into two categories. The ﬁrst research focuses on the establishment of diagnostic criteria for depression. Multiple research has established diagnostic criteria, including scales and grading methods used by physicians, and the diagnosis of MDD has been further strengthened based on these deﬁnitive studies (29–33). The other is the detection of MDD brain regions in radiology methods, such as the cerebral cortex, Large-Scale Network Dysfunction, and the use of inﬂuence studies to study MDD and to determine its correlation and build a scale (34– 36). These highly cited papers have played a vital role in clinical research, diagnosis, and image registration algorithm. These two types of publications will be cited more in the future based on the research results, these studies have found a precise and reproducible research pattern in the complex relationship between MDD and imaging to a certain extent, and the number of citations will be increased in the foreseeable future. mean a more signiﬁcant impact and a link to research. In addition, the number of internal citations by other selected publications is considered to be a critical performance in the ﬁeld. A total of 395 publications were extracted and identiﬁed as reviews. The essential characteristics of the number of publications are shown in Figure 1. The average number of publications is 19.75 per year, and it can be found that the number of publications has increased signiﬁcantly since 2016, and it is expected that the number of papers published in 2022 maybe 80. Table 3 shows the 10 most internally cited reviews and their total citation counts. Among them, Bolay H’s migraine model research is the most cited internally, with 10 internal citations (18), and Julian’s inconsistency in meta- analyses research, with total citations of 36,995. High-cited reviews focus on reviews related to pathogenic mechanisms Review plays a role in summarizing a branch research direction in MDD and radiology research. High citations often Frontiers in Psychiatry 04 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 10.3389/fpsyt.2022.978763 TABLE 2 Inclusive and exclusive criteria. Parameter of selection of a publication Inclusion criterion Exclusion criterion Rationale for inclusion– exclusion Language English All other languages The working language of LDA algorithm is English. Year 2002–2021 Publications before 2021 and after 2002 2022’s publications cannot be included because it has not been fully published Publication Type All Missing data, meeting abstract, proceeding paper, book review, news item In addition to incomplete content, try to include research articles and review Funding sponsor All No exclusion This parameter does not aﬀect the selection criterion Aﬃliation All No exclusion This parameter does not aﬀect the selection criterion Funding All No exclusion This parameter does not aﬀect the selection criterion Country All No exclusion Publication from each country has its signiﬁcance studies based on trials and data may appear in the future to guide research on MDD and radiology-related mechanisms. studies based on trials and data may appear in the future to guide research on MDD and radiology-related mechanisms. Frontiers in Psychiatry Hospitals play an essential role in major depressive disorder and radiology research The organization of a publication can often indicate the publication’s research location and characteristics. Because multiple research institutions often accompany publications. The PubMed database provides the detailed organization of each author of the manuscript. The publications’ ﬁrst organization was analyzed to facilitate statistics and reﬁne the author’s speciﬁc department. The organization that published the most papers was Huaxi MR Research Center from West China Hospital, Sichuan University, China, with 25 publications, followed by the Department of Psychiatry, University of Munster, Munster, Germany, with 12 publications (Table 5). It was found that most research organizations were hospitals, especially radiology High citation review mainly covers the mechanism of acute injury, inﬂammation, genetic factors, trauma, vascular microembolism, ischemia, and migraine model on MDD, and also includes the criteria and discussion of systematic review methods (18–27). It shows that high-cited reviews focus on reviews related to pathogenic mechanisms, and this trend will continue (Figure 2 and Supplementary Figure 1). At the same time, combined with Meta-analysis and the increase in the number of publications of clinical trials, more systematic review 05 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 FIGURE 2 Changes in depression and radiology publication types over the last 20 years. Publication types are deﬁned by the PubMed database and extracted using Python. TABLE 3 Top 10 review publications of major depressive disorder (MDD) and radiology based on internal citations. Reference title DOI Title Internal citation Total citation Nat Med. 2002 Feb;8(2):136-42 10.1038/nm0202-136 Intrinsic brain activity triggers trigeminal meningeal aﬀerents in a migraine model (18) 10 825 BMJ. 2003 Sep 6;327(7414):557-60 10.1136/bmj.327.7414.557 Measuring inconsistency in meta-analyses (19) 9 36,995 J Cereb Blood Flow Metab. 2009 Sep;29(9):1517-27 10.1038/jcbfm.2009.73 Persistent increase in oxygen consumption and impaired neurovascular coupling after spreading depression in rat neocortex (24) 9 151 Nat Med. 2011 Apr;17(4):439-47 10.1038/nm.2333 The role of spreading depression, spreading depolarization and spreading ischemia in neurological disease (26) 9 704 PLoS Med. 2009 Jul 21;6(7):e1000097 10.1371/journal.pmed.1000097 Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement (23) 9 24,250 Ann Neurol. 2010 Feb;67(2):221-9 10.1002/ana.21871 Microemboli may link spreading depression, migraine aura, and patent foramen ovale (25) 8 200 J Cereb Blood Flow Metab. 2011 Jan;31(1):17-35 10.1038/jcbfm.2010.191 Clinical relevance of cortical spreading depression in neurological disorders: migraine, malignant stroke, subarachnoid and intracranial hemorrhage, and traumatic brain injury (27) 8 389 J Clin Invest. Hospitals play an essential role in major depressive disorder and radiology research 2009 Jan;119(1):99-109 10.1172/JCI36059 Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1(21) 8 195 Biol Psychiatry. 2009 May 1;65(9):732-41 10.1016/j.biopsych.2008.11.029 Inﬂammation and its discontents: the role of cytokines in the pathophysiology of major depression (22) 7 2,335 Brain. 2006 Mar;129(Pt 3):778-90 10.1093/brain/awh716 Cortical spreading depression and peri-infarct depolarization in acutely injured human cerebral cortex (20) 7 299 FIGURE 2 Changes in depression and radiology publication types over the last 20 years. Publication types are deﬁned by the PubMed database and extracted using Python. FIGURE 2 Changes in depression and radiology publication types over the last 20 years. Publication types are deﬁned by the PubMed database and extracted using Python. p p j p ( ) gy Reference title DOI Title Internal citation Total citation Nat Med. 2002 Feb;8(2):136-42 10.1038/nm0202-136 Intrinsic brain activity triggers trigeminal meningeal aﬀerents in a migraine model (18) 10 825 BMJ. 2003 Sep 6;327(7414):557-60 10.1136/bmj.327.7414.557 Measuring inconsistency in meta-analyses (19) 9 36,995 J Cereb Blood Flow Metab. 2009 Sep;29(9):1517-27 10.1038/jcbfm.2009.73 Persistent increase in oxygen consumption and impaired neurovascular coupling after spreading depression in rat neocortex (24) 9 151 Nat Med. 2011 Apr;17(4):439-47 10.1038/nm.2333 The role of spreading depression, spreading depolarization and spreading ischemia in neurological disease (26) 9 704 PLoS Med. 2009 Jul 21;6(7):e1000097 10.1371/journal.pmed.1000097 Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement (23) 9 24,250 Ann Neurol. 2010 Feb;67(2):221-9 10.1002/ana.21871 Microemboli may link spreading depression, migraine aura, and patent foramen ovale (25) 8 200 J Cereb Blood Flow Metab. 2011 Jan;31(1):17-35 10.1038/jcbfm.2010.191 Clinical relevance of cortical spreading depression in neurological disorders: migraine, malignant stroke, subarachnoid and intracranial hemorrhage, and traumatic brain injury (27) 8 389 J Clin Invest. 2009 Jan;119(1):99-109 10.1172/JCI36059 Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1(21) 8 195 Biol Psychiatry. 2009 May 1;65(9):732-41 10.1016/j.biopsych.2008.11.029 Inﬂammation and its discontents: the role of cytokines in the pathophysiology of major depression (22) 7 2,335 Brain. 2006 Mar;129(Pt 3):778-90 10.1093/brain/awh716 Cortical spreading depression and peri-infarct depolarization in acutely injured human cerebral cortex (20) 7 299 Reference title 06 Frontiers in Psychiatry frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 TABLE 4 Top 10 cited publications of major depressive disorder (MDD) and radiology. Reference title DOI Title TABLE 4 Top 10 cited publications of major depressive disorder (MDD) and radiology. Reference title DOI Title Internal citation Total citations Neuroimage. 2002 Jan;15(1):273-89 10.1006/nimg.2001.0978 Automated anatomical labeling of activations in SPM using a macroscopic anatomical parcellation of the MNI MRI single-subject brain (28) 109 11,042 Biol Psychiatry. 2007 Sep 1;62(5):429-37 10.1016/j.biopsych.2006.09.020 Resting-state functional connectivity in major depression: abnormally increased contributions from subgenual cingulate cortex and thalamus (50) 84 1,506 JAMA Psychiatry. 2015 Jun;72(6):603-11 10.1001/jamapsychiatry.2015.0071 Large-Scale Network Dysfunction in Major Depressive Disorder: A Meta-analysis of Resting-State Functional Connectivity (36) 76 941 Neuroimage. 2006 Jul 1;31(3):968-80 10.1016/j.neuroimage.2006.01.021 An automated labeling system for subdividing the human cerebral cortex on MRI scans into gyral based regions of interest (35) 68 6,651 Neuron. 2002 Jan 31;33(3):341-55 10.1016/s0896-6273(02)00569-X Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain (34) 68 5,508 Ann N Y Acad Sci. 2008 Mar;1124:1-38 10.1196/annals.1440.011 The brain’s default network: anatomy, function, and relevance to disease (31) 63 6,922 Neuroimage. 2007 Oct 15;38(1):95-113 10.1016/j.neuroimage.2007.07.007 A fast diﬀeomorphic image registration algorithm (30) 62 5,255 Neuroimage. 2012 Feb 1;59(3):2142-54 10.1016/j.neuroimage.2011.10.018 Spurious but systematic correlations in functional connectivity MRI networks arise from subject motion (33) 61 4,631 Neuroimage. 2004;23 Suppl 1:S208-19 10.1016/j.neuroimage.2004.07.051 Advances in functional and structural MR image analysis and implementation as FSL (29) 55 8,671 Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):11020-5 10.1073/pnas.1000446107 Resting-state functional MRI in depression unmasks increased connectivity between networks via the dorsal nexus (32) 55 768 (Figure 3B). The study found that Southeast Asia and Africa contributed less than 5% of the total publications. Based on geographic data, the vast majority of the world’s population participates in relevant research, but there are considerable diﬀerences in published data. departments, accounting for more than 70%. This suggests that a hospital is an essential research organization to study MDD and radiology departments because they are more likely to obtain support from clinical departments and patients, and colleagues in the radiology department can assist in scientiﬁc research, diagnosis, and follow-up. It also suggests that further scientiﬁc research departments, fundamental neuroscience research departments, may be able to conduct more profound research through better cooperation with hospitals. Medical Subject Heading terminology research suggests that “diagnostic imaging,” “MRI,” and “physiopathology” are the most studied areas The United States, China, and Japan have the highest number of publications in the ﬁeld of depression and radiology Symptoms and Sleep are the topics of most signiﬁcant interest in research. As the connecting line indicates, there is a strong relationship between “Breast cancer-related depression” and “Mechanism study,” indicating that the links are closely related. The blue cluster “Brain Structure and Imaging” covers branches including “Brain structure study” and “Functional connectivity,” “Neuroimaging and diagnosis,” “Cognitive impairment,” “Adolescence and depression,” “SSRIs and depression.” This cluster can be interpreted as the study of the depressive brain by multiple imaging techniques. The rose-red cluster “Comorbidities research” includes its branches “Depression and age,” “Case reports,” “Depression with coronary artery disease,” “Fractures and depression,” “HIV and depression,” and other research. This cluster suggests that coronary heart disease, fractures, and lung diseases are closely related to depression and radiology. In conclusion, research on depression and radiology covers a wide range of clinical, brain structures, and comorbidities of depression. TABLE 5 Top 10 organizations with the most publications. Organizations and departments Publication number Rank Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital of Sichuan University, Chengdu, China 25 1 Department of Psychiatry, University of Munster, Munster, Germany 12 2 Pain Relief and Palliative Care Unit, Department of Radiology, Areteion Hospital, School of Medicine, University of Athens, Athens, Greece 10 3 Department of Psychological & Brain Sciences, Washington University in St. Louis, USA 9 4 Department of Psychiatry, Washington University in St. Louis, USA 7 5 Department of Radiology, The Second Xiangya Hospital, Central South University, Changsha, China 7 6 Department of Psychiatry, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada 6 7 Department of Radiology, The First Aﬃliated Hospital of Anhui Medical University, Hefei, China 6 8 Department of Psychiatry, University of Occupational and Environmental Health, Kitakyushu, Japan 5 9 Neurovascular Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA 5 10 Latent Dirichlet allocation results suggest that Symptoms and Sleep, Brain structure study, and functional connectivity are recent research focus Latent Dirichlet allocation results were visualized with a heatmap to understand further the evolution of the general research focus in depression and radiology. The heatmap shows 50 research topics and annual changes in publications from 2002 to 2021 (Figure 6). Publication numbers in the three areas of Symptoms and Sleep, Brain structure study, and functional connectivity have increased dramatically. Therefore, based on the number of publications, these three topics may be the focus of current and future research. Hippocampus are the most studied. Furthermore, the diﬀerence in the number of studies on other structures is not apparent, but in terms of the number of studies, these structures may be the most closely related to depression and radiology and suggest that they are the most concerned by researchers (Figure 4C). Latent Dirichlet allocation identiﬁed the main areas of depression and radiology are“Symptoms and Treatment,” ”Brain structure and Imaging,” and ”Comorbidities research” The United States, China, and Japan have the highest number of publications in the ﬁeld of depression and radiology To further explore the changes in research directions, the MeSH terms of all publications were extracted and analyzed, which can partially reﬂect research topics. 5,561 MeSH were studied, and all MeSH terms have been studied 85,181 times, indicating that the research scope of depression and radiology is extensive. Diagnostic imaging, magnetic resonance imaging, and physiopathology have been studied the most, reaching 1156, 802, and 632 times (Figure 4A). Interestingly, 2,153 and 1,618 studies on the Adult and Aged populations in age-related MeSH terms were found, indicating that researchers conducted the most studies on adults (Figure 4B). Regarding brain structure, 10 MeSH terms with the most research on brain structure were extracted, among which Cerebral Cortex, Prefrontal Cortex, and Next, the author’s aﬃliation information was analyzed to study research institutions’ geographic distribution. Institutions in 76 countries or regions worldwide were identiﬁed, most located in the northern hemisphere, participated in relevant research and published manuscripts (Figure 3A). Among all publications, the USA, China, Japan, Germany, and the Netherlands accounted for 21.5, 20.6, 6.4, 5.4, and 4.6%, respectively. The top 10 countries with the most signiﬁcant publications accounted for 73.9% of all publications Frontiers in Psychiatry 07 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 10.3389/fpsyt.2022.978763 "Symptoms and Treatment" can be subdivided into “Symptoms and Sleep,” “Treatment trial,” “Treatment trial,” “Review,” and other areas, with the circle size determined by the number of publications. Symptoms and Sleep are the topics of most signiﬁcant interest in research. As the connecting line indicates, there is a strong relationship between “Breast cancer-related depression” and “Mechanism study,” indicating that the links are closely related. The blue cluster “Brain Structure and Imaging” covers branches including “Brain structure study” and “Functional connectivity,” “Neuroimaging and diagnosis,” “Cognitive impairment,” “Adolescence and depression,” “SSRIs and depression.” This cluster can be interpreted as the study of the depressive brain by multiple imaging techniques. The rose-red cluster “Comorbidities research” includes its branches “Depression and age,” “Case reports,” “Depression with coronary artery disease,” “Fractures and depression,” “HIV and depression,” and other research. This cluster suggests that coronary heart disease, fractures, and lung diseases are closely related to depression and radiology. In conclusion, research on depression and radiology covers a wide range of clinical, brain structures, and comorbidities of depression. "Symptoms and Treatment" can be subdivided into “Symptoms and Sleep,” “Treatment trial,” “Treatment trial,” “Review,” and other areas, with the circle size determined by the number of publications. Frontiers in Psychiatry Discussion Machine learning and NLP were used to analyze 5,566 PubMed publications in depression radiology research from 2002 to 2022. The results from multiple perspectives were analyzed and visualized, including publication numbers, geographic information, MeSH terms, and topic model algorithms. This is the ﬁrst such analysis in this ﬁeld. Over the past 20 years, the number of depression and radiology research publications has increased from 3,359 in 2002 to 10,145 in 2021. The top-cited publications were 11,042, and the highly-cited publications focused on improving diagnostic performance and establishing imaging standards. Hospitals and radiology departments take the lead in research and have an advantage. To further identify a speciﬁc research focus, the topic model LDA algorithm were constructed to generate relevant topics based on the content of each publication. To express the relationships between interrelated topic clusters and underlying topics, topic network analysis and visualization analysis using LDA and Leuven algorithms were built. The algorithm divides the past 20 years of research into these three research clusters, “Symptoms and Treatment,” “Brain Structure and Imaging,” and “concurrent disease research” (Figure 5). The line thickness between the circles indicates the correlation between the two topics. For example, the orange marker cluster Frontiers in Psychiatry 08 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 FIGURE 3 Regional differences in the research status of global depression and radiology. (A) The map shows the global distribution of depression and radiology publications over 20 years. Country information was extracted based on publication afﬁliation. Countries or regions were drowned with publications; the darker the color, the greater the number of publications. The number of publications in the Northern Hemisphere is much higher than in the Southern Hemisphere. (B) Top 15 countries with the most published depression and radiology studies. FIGURE 3 Regional differences in the research status of global depression and radiology. (A) The map shows the global distribution of depression and radiology publications over 20 years. Country information was extracted based on publication afﬁliation. Countries or regions were drowned with publications; the darker the color, the greater the number of publications. The number of publications in the Northern Hemisphere is much higher than in the Southern Hemisphere. (B) Top 15 countries with the most published depression and radiology studies. The extensive ﬁeld of study contains 12,058 MeSH terms. Many regions worldwide participated in the study, especially the Northern Hemisphere. Frontiers in Psychiatry Discussion In the past 20 years, depression radiology research has mainly focused on “Symptoms and Treatment,” “Brain Structure and Imaging,” and “Comorbidities research.” Signiﬁcantly the research focus has changed in recent years, and the current research focus is expected to be on symptoms and sleep, brain structure study, and functional connectivity. analyzed text and can be used on personal computers. At the same time, multiple visualization software and the opinions of multiple professional authors were combined, striving to be able to explain the machine learning results. In addition, bibliometric analysis can be performed with a few software, such as Bibliographic Items Co-Occurrence Matrix Builder (BICOMB), VOSviewer, and CiteSpace (37, 38). However, these software cannot analyze a large amount of text processing, so a new analysis method was built. Machine learning and NLP often analyze large amounts of data resulting in diﬃculty understanding the results, also known as inexplicability. The LDA algorithm was used, an unsupervised algorithm that does not require labels and training sets for the This study found that the radiology study of depression includes many studies on the comorbidities of depression, such as breast cancer, fracture, coronary artery disease, Alzheimer’s disease (AD), and respiratory system diseases, accounting for 09 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 FIGURE 4 Research changes in depression and radiology through a study of MeSH terminology. R extracted signiﬁcant MeSH terms from publications listed in the PubMed database according to the publication topic. Each publication is deﬁned by the PubMed database, which can indicate the general content of the paper research. Each publication contains multiple MeSH terms, which extracted by R and Python. (A) Ten of the most widely studied MeSH terms and their number (No.) of publications per year. “diagnostic imaging,” “MRI,” and “physiopathology” were the most frequently studied research ﬁelds in the last 20 years. (B) The number of publications studying different age groups. Adults is the most studied group. (C) The most studied brain structures and their number of publications. The Cerebral Cortex, Prefrontal Cortex, and Hippocampus are the most studied brain structures. FIGURE 4 Research changes in depression and radiology through a study of MeSH terminology. R extracted signiﬁcant MeSH terms from publications listed in the PubMed database according to the publication topic. Each publication is deﬁned by the PubMed database, which can indicate the general content of the paper research. Each publication contains multiple MeSH terms, which extracted by R and Python. Discussion (A) Ten of the most widely studied MeSH terms and their number (No.) of publications per year. “diagnostic imaging,” “MRI,” and “physiopathology” were the most frequently studied research ﬁelds in the last 20 years. (B) The number of publications studying different age groups. Adults is the most studied group. (C) The most studied brain structures and their number of publications. The Cerebral Cortex, Prefrontal Cortex, and Hippocampus are the most studied brain structures. about 20% of the total publications. About 668 publications are Case Reports. On the one hand, many patients cannot accurately identify their depression. About 50–70% of patients are found to suﬀer from depression because of comorbidities (39). Many studies have pointed out that depression, coronary artery disease, and diabetes have mutually reinforcing risks (40, 41). On the other hand, these patients often undergo various radiological examinations to identify lesions and assess disease status, which has increased people’s understanding of depression from another perspective. Depression can be observed in 50% of AD patients, and MRI revealed widespread brain atrophy and disruption of white matter integrity and cognitive impairment, and higher depression severity (42). this study found breast cancer; a signiﬁcant correlation with Frontiers in Psychiatry Frontiers in Psychiatry 10 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 FIGURE 5 Latent Dirichlet allocation (LDA) identiﬁed the main areas of depression and radiology are “Symptoms and Treatment,” ”Brain Structure and Imaging,” and ”Comorbidities research.” The LDA algorithm was used to analyze publications from the last 20 years. from which 50 topics were identiﬁed and classiﬁed into three broad categories: “Symptoms and Treatment” (orange), ”Brain Structure and Imaging” (blue), and ”Comorbidities research” (rose-red). The circle size represents the number of publications contained in the research topic. For example, the algorithm found that the topic “Depression and age” contains 395 publications. The line thickness between the circles represents the degree of overlap between the two research topics, such as the 148 publications with the highest overlap in “Breast cancer-related depression” and “Mechanism study.” FIGURE 5 Latent Dirichlet allocation (LDA) identiﬁed the main areas of depression and radiology are “Symptoms and Treatment,” ”Brain Structure and Imaging,” and ”Comorbidities research.” The LDA algorithm was used to analyze publications from the last 20 years. from which 50 topics were identiﬁed and classiﬁed into three broad categories: “Symptoms and Treatment” (orange), ”Brain Structure and Imaging” (blue), and ”Comorbidities research” (rose-red). Discussion The circle size represents the number of publications contained in the research topic. For example, the algorithm found that the topic “Depression and age” contains 395 publications. The line thickness between the circles represents the degree of overlap between the two research topics, such as the 148 publications with the highest overlap in “Breast cancer-related depression” and “Mechanism study.” breast cancer patients are more vulnerable to depression (43). In conclusion, depression and comorbidities can promote the study of depression by imaging from another perspective. studies on mechanisms of depression, with a weight of 148. In the further analysis, results shows found that there is a strong correlation between “women,” “age,” “lack of sleep,” and “COVID-19.” Breast cancer radiology studies uncover most underlying mechanisms underlying a functional dorsal attention network (DAN). Changes in breast cancer patients without chemotherapy may be due to insuﬃcient frontoparietal neural activity to drive DAN and may be related to the eﬀects of neuropsychological distress. Suggesting that older women and The brain structure study lists the top 10 most studied brain structures in depression radiology in the last 20 years. The top three are the cerebral cortex, prefrontal cortex, and hippocampus, with 232, 201, and 182 publications. This may mean that researchers have the most understanding of the brain structure of depression from a radiology perspective. Based on Frontiers in Psychiatry 11 frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 FIGURE 6 “Symptoms and sleep,” “Brain structure study,” and “functional connectivity.” The heatmap presents the change of 50 research topics over the past 20 years. Data were generated by the use of the LDA algorithm. The abscissa represents the year, the ordinate represents the topics, and the color brightness represents the number of publications and reﬂects the shift in research focus. The legend represents the number of publications per color. FIGURE 6 “Symptoms and sleep,” “Brain structure study,” and “functional connectivity.” The heatmap presents the change of 50 research topics over the past 20 years. Data were generated by the use of the LDA algorithm. The abscissa represents the year, the ordinate represents the topics, and the color brightness represents the number of publications and reﬂects the shift in research focus. The legend represents the number of publications per color. “Symptoms and sleep,” “Brain structure study,” and “functional connectivity.” The heatmap presents the change of 50 research topics over the past 20 years. Funding This study was supported by the National Nature Scientiﬁc Foundation of China (Grant No. 81702956), the Strategy- Oriented Special Project of Central South University in China (Grant No. ZLXD2017003), the Natural Science Foundation of Hunan Province (Grant Nos. 2020JJ4903, 2020JJ5920, and 2020JJ4883 to LK), and the Colorectal cancer medical seed research fund project named “Eﬀect and mechanism of YAP1 on EGFR resistance in K-ras wild-type metastatic colorectal cancer” from the Beijing Bethune Public Welfare Foundation. As for the limitations, ﬁrstly, all the included publications are from the PubMed database, and the publications only include English papers. The data included in PubMed is relatively complete; it may still be biased (49). Secondly, road search terms were used to include the most comprehensive publications possible, which may lead to bias. This study uses an analytical approach from an NLP perspective and superﬁcial Discussion Data were generated by the use of the LDA algorithm. The abscissa represents the year, the ordinate represents the topics, and the color brightness represents the number of publications and reﬂects the shift in research focus. The legend represents the number of publications per color. “Symptoms and sleep,” “Brain structure study,” and “functional connectivity.” The heatmap presents the change of 50 research topics over the past 20 years. Data were generated by the use of the LDA algorithm. The abscissa represents the year, the ordinate represents the topics, and the color brightness represents the number of publications and reﬂects the shift in research focus. The legend represents the number of publications per color. publications exceeded 300, and the number of publications in recent years has shown a clear upward trend. This shows that researchers’ understanding of depression from a radiological perspective begins to deepen the connection between brain providing data from 10 brain lesions, building a scoring system based on big data in radiology may be a new way to assess depression (44). In addition, LDA analysis found that under the research topic of functional connectivity, the number of Frontiers in Psychiatry 12 Frontiers in Psychiatry frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 10.3389/fpsyt.2022.978763 insights into depression and radiology research topics. However, machine learning and NLP are new tools for scientists to extract objective and comprehensive clues from large amounts of data. regions. A recent study using machine learning found that the co-activation pattern (CAP) of the salience network (SN) in MDD patients has a transitional enhancement to CAP (45). The results showed that the ﬂexibility of the network was enhanced in patients. The study also found reduced spatial consistency and persistence of the default mode network (DMN) in patients, suggesting reduced variability and stability in patients with MDD (46). Based on this, it is speculate that this direction will be the focus of research in the future, and it is very likely to combine brain metabolomics and brain function to discover new critical therapeutic targets. Conclusion Latent Dirichlet allocation analysis methods can be well used to analyze many texts and discover recent research trends and focus. In the past 20 years, the research on MDD and radiology has focused on exploring MDD mechanisms, establishing standards, and constructing imaging methods. Recent research focuses are “Symptoms and sleep,” “Brain structure study,” and “functional connectivity.” New progress may be made in studies on MDD complications and the combination of brain structure and metabolism. Radiology is now widely used to diagnose, treat, evaluate, and research MDD. More than 400 clinical trials of various types have been published, covering a variety of diseases, such as tumors, and radiological evaluation of the treatment of post-traumatic depression. There are currently more than 400 publications related to clinical trials, covering a variety of diseases, such as cancer, and radiological evaluation of the treatment of post-traumatic depression. However, many keywords related to primary medical institutions and related research topics, Caregiver and Primary care, were found in the diagnosis of MDD. Harman’s study shows that adults with clinically signiﬁcant unipolar depression tend to present to primary care rather than psychiatry, and 50% were not screened or evaluated for depression (47, 48). Recent studies have identiﬁed a potential link between connectome gradient disruption and the sensory-cognitive impairment accompanying MDD in MDD patients from the functional connectome and DMN. With the aid of various new radiological tools such as fMRI and MRS for diagnosis, better radiological diagnostic and predictive criteria will be constructed for the medication response of MDD patients. Data availability statement The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Author contributions KW collected and analyzed the data and wrote the manuscript. FT, ZZ, and LK provided extensive guidance and feedback on the manuscript. All authors contributed to the design of the study and have read and approved the ﬁnal manuscript. In addition, sleep disorders and sleep-wake disorders as MeSH terms appeared in more than 500 publications. Studies have found that more than 70% of MDD patients suﬀer from sleep disturbances. Radiology plays a vital role in sleep and MDD-related research—especially the application of recent radiology technology. The current study conﬁrmed that amygdala-based rtfMRI-NF training altered intrinsic functional centers remodeled abnormal functional connectivity caused by insomnia, and improved sleep in CID patients. These ﬁndings contribute to understanding of the neurobiological mechanisms of rtfMRI-NF in the treatment of insomnia (8). There has been a rapid increase in the number of papers published in this ﬁeld in recent years, and there may be new advances in research predicting new technologies for sleep disorder research. Frontiers in Psychiatry Acknowledgments We want to express our gratitude to Wen Yan, who supported the study by programming. 13 frontiersin.org frontiersin.org Wang et al. 10.3389/fpsyt.2022.978763 Publisher’s note The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fpsyt.2022.978763/full#supplementary-material All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated Conﬂict of interest 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. 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. References 16. Blei DM, Ng AY, Jordan MI. Latent dirichlet allocation. J Mach Learn Res. 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https://openalex.org/W4283696668	https://www.frontiersin.org/articles/10.3389/fendo.2022.904986/pdf	English	null	Hürthle Cell Carcinoma: Single Center Analysis and Considerations for Surgical Management Based on the Recent Literature	Frontiers in endocrinology	2,022	cc-by	5,203	Costanza Chiapponi 1, Milan J.M. Hartmann 1, Matthias Schmidt 2, Michael Faust 3, Christiane J. Bruns 1, Anne M. Schultheis 4 and Hakan Alakus 1 1 Department of General, Visceral, Cancer and Transplant Surgery, University Hospital Cologne, Cologne, Germany, 2 Department of Nuclear Medicine, Faculty of Medicine, University Hospital Cologne and University Hospital Cologne, Cologne, Germany, 3 Polyclinic for Endocrinology, Diabetes and Preventive Medicine, University Hospital Cologne, Cologne, Germany, 4 Department of Pathology, University Hospital Cologne, Cologne, Germany Background: Hürthle cell carcinoma (HCC) of the thyroid is rare. There are contrasting data on its clinical behavior. The aim of this study was to describe clinic-pathological features and outcomes of HCC patients at our institution, in order to adapt our surgical management. Background: Hürthle cell carcinoma (HCC) of the thyroid is rare. There are contrasting data on its clinical behavior. The aim of this study was to describe clinic-pathological features and outcomes of HCC patients at our institution, in order to adapt our surgical management. ORIGINAL RESEARCH published: 29 June 2022 doi: 10.3389/fendo.2022.904986 ORIGINAL RESEARCH published: 29 June 2022 doi: 10.3389/fendo.2022.904986 Keywords: Hürthle cell carcinoma, thyroid cancer, prophylactic lymphadenectomy, lymph node involvement, recurrence of HCC Edited by: Trevor Edmund Angell, University of Southern California, United States outcomes of HCC patients at our institution, in order to adapt our surgical management. Methods: We retrospectively studied 51 cases of HCC treated at the interdisciplinary endocrine center of the University Hospital of Cologne, Germany between 2005 and 2020. Results: Patients median age was 63 years (range 29-78) with 64.7% of cases being female. Primary treatment included surgery and postoperative radioiodine therapy with 3.7 GBq in all patients. Surgery consisted of total thyroidectomy in all cases and additional central lymphadenectomy in 90.2% of cases. The median number of harvested lymph nodes was 11 (range 2-31). Lymph node involvement was found in two (4.3%) pT4a tumors. In all other cases (95.7%), central lymphadenectomy was prophylactic and lymph nodes were free of metastasis in ﬁnal histopathology. Twelve (23.5%) patients with incomplete biochemical response to primary treatment were diagnosed with structural relapse during the course of disease, for which seven (58.4%) underwent resection of isolated cervical metastasis. Histopathology revealed soft tissue implants in all cases and cervical surgery led to biochemical and radiologic cure in only two (28.5%) cases. Five (41.6%) patients developed metastatic disease, followed by systemic therapy in two patients. Vascular invasion of the primary tumor was signiﬁcantly associated with relapse (p<0.01). Methods: We retrospectively studied 51 cases of HCC treated at the interdisciplinary endocrine center of the University Hospital of Cologne, Germany between 2005 and 2020. Reviewed by: Jeffrey A Knauf, Cleveland Clinic, United States James Wu, University of California, Los Angeles, United States University of California, Los Angeles, United States Correspondence: Costanza Chiapponi Costanza.Chiapponi@uk-koeln.de Specialty section: This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology Received: 26 March 2022 Accepted: 16 May 2022 Published: 29 June 2022 Hürthle Cell Carcinoma: Single Center Analysis and Considerations for Surgical Management Based on the Recent Literature Costanza Chiapponi 1*, Milan J.M. Hartmann 1, Matthias Schmidt 2, Michael Faust 3, Christiane J. Bruns 1, Anne M. Schultheis 4 and Hakan Alakus 1 Primary Treatment According to the current German Guidelines (10), primary surgical management for pre- or intraoperatively diagnosed HCC consists of total thyroidectomy and prophylactic central lymphadenectomy. For postoperatively diagnosed HCC two- stage thyroidectomy is recommended. For patients with still elevated thyroglobulin levels or suspicious lymph nodes after total thyroidectomy, lymphadenectomy is suggested (10). RAIT is recommended for all patients. At our institution diagnostic whole-body scintigraphy scan (DWBS) is regularly performed 6-9 months after RAIT and represents the only staging parameter routinely performed for differentiated thyroid cancer, besides comprehensive cervical sonography. SPECT/CT of the neck and chest completes the staging. According to the German Guidelines (10), a routine prophylactic central lymph node dissection is recommended in case of preoperative or intraoperative HCC diagnosis. This recommendation is explained by high rates of lymph nodal involvement and impaired iodine avidity of HCC. In case of postoperative diagnosis, prophylactic central lymphadenectomy is recommended for biochemical residual disease, consisting in elevated thyroglobulin level (Tg). These recommendations are based on seven studies (11–17): two reviews (12, 16) and four retrospective analyses (11, 13, 15, 17). They include Lopez et al. (11) reporting a 25% lymph node involvement and a 40% mortality rate in 89 patients, Kushchayeva et al. (13) with 3% lymph node metastasis at presentation and 13% in follow up in 33 patients and Guerrero et al. (17) with lymph node involvement in only 3 (8%) of their 39 patients and in no tumor <5cm. The fourth study quoted in the guidelines is Besic et al. (15). It describes 30.9% of lymph node involvement and 45% of extrathyroidal extension in 42 patients. However, these patients were diagnosed with papillary Hürthle cell carcinoma, which is a variant of papillary thyroid cancer. Citation: Conclusions: Recurrence of HCC was common in this study. Given the low rate of lymph node metastases both in this study and in recent literature and the nature of relapse (soft tissue instead of nodal metastasis), the beneﬁt of routine prophylactic central lymph node dissection for HCC remains unclear, especially in the absence of vascular invasion from the primary tumor. Chiapponi C, Hartmann MJM, Schmidt M, Faust M, Bruns CJ, Schultheis AM and Alakus H (2022) Hürthle Cell Carcinoma: Single Center Analysis and Considerations for Surgical Management Based on the Recent Literature. Front. Endocrinol. 13:904986. doi: 10.3389/fendo.2022.904986 June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org Prophylactic Lymphadenectomy in HCC Chiapponi et al. Surgery of Recurrence Indication to surgery of recurrence in our institution is always initiated by the multidisciplinary endocrine team (MDET). Surgery consists in resection of isolated soft tissue tumors in the perithyroidal/paratracheal space, in case structural recurrence does not appear iodine avid in DWBS or is deemed too large for repeated radioiodine therapy. Surgery is always performed by a specialized endocrine surgeon with intraoperative frozen section and neuromonitoring (19). Patients and Tumors Patients, who underwent radioiodine treatment (RAIT) between 01.01.2005 and 31.07.2020 at the University Hospital of Cologne, Germany for differentiated thyroid cancer, were screened. The group of Hürthle cell carcinomas (HCC) according to the current WHO deﬁnition was identiﬁed [more than 75% of the tumor consists of Hürthle cells (18)]. Papillary Hürthle cell carcinoma is a distinct entity and was not included in the present study. INTRODUCTION Patients receiving surgical treatment at our institution as well as those referred to our center for RAIT and oncologic care after external surgery were included in the present study. Hürthle cell carcinoma (HCC) of the thyroid is rare. It is reported to account for only 3-4% of all thyroid tumors (1). A recent retrospective analysis of HCC primarily treated with surgery at Memorial Sloan Kettering Cancer Center between 1986 and 2015 identiﬁed a total of 111 patients only (2). Histologic classiﬁcation was made according to the at the time current IACR WHO Classiﬁcation of Tumors of Endocrine Organs, representing Hürthle cell carcinoma in the current 2017, 4th edition of the WHO Classiﬁcation of Tumors of Endocrine Organs. Staging was performed using the UICC TNM classiﬁcation of malignant tumors (18). HCC was previously classiﬁed as a variant of follicular thyroid carcinoma but was reclassiﬁed in 2017 in the 4th WHO classiﬁcation of Tumors of Endocrine Organs as a separate subtype of differentiated thyroid cancer (3), due to its distinct genetic expression proﬁle, pathologic characteristics, and clinical behavior. Higher frequencies of distant metastasis, impaired iodine-avidity, and higher rates of mortality have been reported (4, 5), but data are controversial (6–8). Recently, Jin et al. (1) reported persistent/recurrent disease in only 8.2% of the patients, and none of their patients died of HCC during a median follow-up of 8.5 years. Similarly, Oluic et al. (9) reported a rate of 12% of persistence/recurrence, with a slightly worse survival rate than Jin et al. Diagnosis and Radioiodine Treatment for Recurrent Disease Diagnosis of recurrence was based on physical examination, thyroglobulin (Tg) increment and imaging studies. During follow up, signiﬁcant elevation of basal and stimulated serum Tg compared to the nadir value as well as all values >1 ng/ mL measured with an ultrasensitive assay led to a diagnostic whole-body scintigraphy scan (DWBS) with 185-370 MBq of radioiodine, followed by 18F-FDG PET-CT if radioiodine uptake was low or absent. If DWBS studies were positive, patients received a therapeutic activity of 3.7 of radioiodine (I-131). If DWBS was negative, but 18F-FDG PET-CT conﬁrmed structural recurrence, surgery was considered. If the imaging studies did not display recurrence, serum TG levels were monitored, as described previously (19). Given the conﬂicting data in the literature, we aimed at investigating clinic-pathological features and outcomes of HCC patients treated at our institution, in order to critically evaluate surgical management of these patients. Repeated Cervical Surgery Among 1897 patients receiving radioiodine treatment (RAIT) for differentiated thyroid cancer, 51 (2.7%) were diagnosed with HCC (Table 1). Their median age was 63 years (range 29-78). Eighteen (35.3%) patients were male and had a median age of 57 (range 29-76). 33 (64.7%) patients were female and were in median 64 (range 29-78) years old. There was no signiﬁcant gender speciﬁc difference in their age (p=0.27). Second-line surgery for isolated cervical relapse was required in 7 (13.7%) of 51 patients. 71.4% of these patients were male. Their primary tumors consisted of 4 (57.1%) pT2 and 3 (42.9%) pT3 status with vascular invasion (V1/2) in 5 (83.4%) of six pathology reports with V-status documentation. None of them had radiologic (28.5% cN0) or pathologic (71.4% pN0) lymph node involvement at the time of diagnosis. In all cases, initial postoperative radioiodine treatment had resulted in an incomplete biochemical response. Table 2 summarizes the clinic pathological features and outcome of these cases. Follow-Up Follow-up examinations take place every 6 months for 5 years and thereafter yearly in the department for Nuclear Medicine and include physical examinations, cervical ultrasound and laboratory values including thyroglobulin. DWBS, MRI and/or 18FDG PET-CT are planned if necessary. Follow up June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org 2 Prophylactic Lymphadenectomy in HCC Chiapponi et al. examinations until March 2022 were included in this study. Mean follow up was 62 ± 55.9 months after thyroidectomy. Response was evaluated according to the RECIST criteria. of 46 cases lymph node metastases were suspected on preoperative radiologic examination and conﬁrmed in ﬁnal pathologic exam. Both patients had pT4a status. Distant metastases were present in 2 (3.9%) patients at diagnosis, both with pT4a tumors. One of these two patients had no lymph node involvement. Data Collection and Analysis Electronic and paper data of the University Hospital of Cologne were retrospectively collected and analyzed. The study was performed according to the rules and regulations for retrospective analysis of the ethical committee of the University Hospital Cologne. Data were analyzed using IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY. Variables were expressed as median with range. If the data was normally distributed, groups were compared using the T-test. The Chi-square test of independence was used for testing hypotheses when the variables were nominal. A p-value < 0.05 was considered signiﬁcant. V-status was documented in 42 (82.3%) pathology reports and was positive (V1/V2) in 21 (50%) cases, including 3 (30%) of 10 pT1, 9 (34.6%) of 26 pT2, 6 (54.5%) of 11 pT3 and 3 (75%) of 4 pT4 tumors. Radioiodine Treatment All patients received at least one course of radioiodine therapy. In forty-one (80.4%) cases only one course with an activity of 3.7 GBq 131I was administered. In four (7.8%) patients a higher initial dose (5.5 GBq) and in six (11.7%) patients more than one radioiodine treatment were administered during the course of disease, with a median cumulative dose of 7.4 GBq (range 5-55 GBq) 131I. Tumor Stages at the Time of HCC Diagnosis Ten (19.6%) patients had pT1b tumors, 26 (51%) had pT2, 11 (21.6%) had pT3 and 4 (7.8%) had pT4 status. Central lymphadenectomy was performed in 46 (90.2%) patients and delivered a median of 11 lymph nodes (range 2-31). In 2 (4.3%) Surgery was performed, when structural radioiodine refractory disease was diagnosed during the course of disease (Figure 1). In all cases ﬁnal pathology of relapse revealed soft tissue metastases: three paratracheal implants in the central compartment, three jugular tumors in the lateral compartment, one supraclavicular tumor ventral of the sternocleidomastoid muscle. There were no metastatic lymph nodes. Figure 2 shows how a well circumscribed tumor nodule partially surrounded by a ﬁbrous capsule can mimic lymph node metastasis in radiologic studies (Figure 2). TABLE 1 \| Demographic and tumor characteristics of HCC patients included in the present study. N= 51 Age (median, range) 63 (29-78) Gender Male Female 18 (35.3%) 33 (64.7%) pT status pT1 pT2 pT3 pT4 10 (19.6%) 26 (51%) 11 (21.6%) 4 (7.8%) pN status pN0 pN1 pNx 44 (86.2%) 2 (3.9%) 5 (9.8%) pM status pM1 2 (3.9%) V status V0 V1/2 Vx 21 (41.2%) 21 (41.2%) 9 (17.6%) TABLE 1 \| Demographic and tumor characteristics of HCC patients included in the present study. N= 51 Age (median, range) 63 (29-78) Gender Male Female 18 (35.3%) 33 (64.7%) pT status pT1 pT2 pT3 pT4 10 (19.6%) 26 (51%) 11 (21.6%) 4 (7.8%) pN status pN0 pN1 pNx 44 (86.2%) 2 (3.9%) 5 (9.8%) pM status pM1 2 (3.9%) V status V0 V1/2 Vx 21 (41.2%) 21 (41.2%) 9 (17.6%) TABLE 1 \| Demographic and tumor characteristics of HCC patients included in the present study. Overall Outcome After primary treatment, an excellent response was recorded in 38 (72.5%) patients. One (1.96%) patient had an indeterminate response. In 12 (70.6%) patients with initial incomplete biochemical response, structural recurrence was diagnosed during the course of disease. Seven (58.3%) patients with structural recurrence displayed isolated cervical tumors, which were removed surgically (Table 1). Two of these patients displayed an excellent response at last follow up (28.6%). Five (71.4%) presented an indeterminate (n=2), biochemical incomplete (n=2) or structural incomplete response at last follow up (n=1, stable lung metastases in a follow up of 68 months). June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org 3 Prophylactic Lymphadenectomy in HCC Chiapponi et al. TABLE 2 \| Clinic-pathological features of patients undergoing second-line surgery for isolated cervical relapse without distant metastasis. Pt number Gender, Age pT pN cM V Cumulative RAI activity (GBq) Follow up (months) End result (response) • excellent = ER • indeterminate = IR • biochemical incomplete = BI • structural incomplete = SI 1. M, 56 pT2 Nx 0 1 3.7 135 ER 2. M, 74 pT3a 0/17 0 0 3.7 59 BI 3. M, 56 pT3a 0/2 0 1 3.7 42 IR 4. M, 38 pT3a 0/7 0 2 3.7 36 BI 5. F, 59 pT2 0/7 0 1 5.5 211 IR 6. F, 72 pT2 Nx 0 1 7.4 174 ER 7. M, 76 pT2 0/10 0 1 3.7 68 SI Table 2 summarizes the clinic-pathological features of patients undergoing second-line surgery for isolated cervical relapse without distant metastasis. Tg, Thyroglobulin. None of these patients had pathologic Tg antibodies. V= angioinvasions (V0= absent, V1= microscopic, V2= macroscopic). End results were classiﬁed in excellent, indeterminate, biochemical incomplete and structural incomplete response according to Pitoia and Jerkovich (20). FIGURE 1 \| Isolated cervical FDG positive relapse in four patients removed by surgery. FIGURE 1 \| Isolated cervical FDG positive relapse in four patients removed by surgery. FIGURE 1 \| Isolated cervical FDG positive relapse in four patients removed by surgery. Frontiers in Endocrinology \| www.frontiersin.org June 2022 \| Volume 13 \| Article 904986 DISCUSSION Five (41.7%) patients with structural persistence/ recurrence were diagnosed (n=2, 40%) or developed (n=3, 60%) distant metastases in the course of disease and did not receive a second cervical procedure after primary thyroidectomy. Their metastases consisted of pulmonary and/or bone lesions. These patients were older than 65 years (median age 69, range 29-77), with exception of one 29 year old female patient. They were offered tyrosine kinase inhibitors as a systemic treatment but refused in three cases. In both patients receiving systemic treatment, therapy included lenvatinib in ﬁrst line, followed by nivolumab and pembrolizumab, due to side effects of lenvatinib. Both patients discontinued systemic treatment and died two and four years after thyroidectomy respectively. Both patients had widely invasive pT4a tumors and were 66 and 69 years old at the time of diagnosis. The other three experienced slow disease progression. In the present study, the clinic-pathologic features and clinical course of 51 patients treated at our institution in a period of 15 years were analyzed. Despite initial surgery and radioiodine treatment, recurrence rate was as high as 23.5%, which is similar to that previously reported in our patients with follicular thyroid cancer (21) but higher than that reported in some recent literature (1, 2). The reasons underlying these differences are unclear and might be of demographic, ethnic or environmental nature. There is an increasing number of recent studies questioning the reported aggressiveness of HCC. Jin et al. (1) reported persistence/recurrence rates of 8% and no death in a follow up of 8.5 years in South Korea, a region with a good iodine intake. Matsuura et al. (2) compared HCC to FTC in a US American population and found that HCC patients were more likely to recur, but they had shown no signiﬁcant different overall survival. Vascular invasion was a critical prognostic parameter Recurrence was signiﬁcantly more common in tumors, in which vascular invasion (V1 and 2) had been documented (p<0.01). June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org 4 Prophylactic Lymphadenectomy in HCC Chiapponi et al. FIGURE 2 \| (A) Soft tissue implant showing a well-circumscribed tumor nodule partially surrounded by a ﬁbrous capsule clinically mimicking lymph node metastasis. (B) Oncocytic tumor cells form small follicles, sharply separated from surrounding soft tissue. No lymphatic tissue detectable. (C) Tumor free lymph node next to the left jugular vein. DISCUSSION (D) Exemplary clear lymph node from the central compartment in the same patient. FIGURE 2 \| (A) Soft tissue implant showing a well-circumscribed tumor nodule partially surrounded by a ﬁbrous capsule clinically mimicking lymph node metastasis. (B) Oncocytic tumor cells form small follicles, sharply separated from surrounding soft tissue. No lymphatic tissue detectable. (C) Tumor free lymph node next to the left jugular vein. (D) Exemplary clear lymph node from the central compartment in the same patient. for predicting recurrence in their study (2). The current German Guidelines recommend total thyroidectomy and routine prophylactic central lymphadenectomy for Hürthle cell carcinoma irrespective of V status (10). tissue or capsule in most cases (25). This important aspect is probably not given enough clinical attention. These data together with those of Coca Pelaz et al. (26), suggest that prophylactic central lymphadenectomy might not be of beneﬁt in HCC. In their meta-analysis Coca Pelaz et al. included 9638 patients and found lymph node metastasis in only 9% of patients (26). Due to the impaired iodine avidity of HCC (22) and the assumed high rate of lymph node involvement, the guidelines recommend prophylactic central lymphadenectomy (10). Accordingly, this was regularly performed (90.2% of cases). However, despite a sufﬁcient median number of harvested lymph nodes (n=11), lymph node metastases were found in only two (4.3%) patients presenting with pT4a status. This rate is similar to that of Goffredo et al. (23), who documented node-positive disease in 5.3% of cases. The only two studies reporting a high nodal involvement are Chindris et al. (24) (21.9%) and Lopez et al. (11). However, it needs to be mentioned that in Chindris et al. lymph nodes were not examined in the majority of cases (57.8%), therefore the rate of lymph node involvement is probably not representative. The collective of Lopez et al. (11) includes 7 tumors with anaplastic foci, which might inﬂuence nodal involvement rates. Soft tissue metastatic nodules can generally be identiﬁed in preoperative 18F-FDG PET scan (Figure 1). However, surgeons should be aware of the presence of additional sub centimeter foci, which might sometimes be found only by accurate palpation and intraoperative sonography and sometimes are even missed. In fact, only 2 (28.6%) of the patients undergoing cervical salvage surgery in the present analysis could be cured. All others had at least biochemical residual disease (Table 2). Frontiers in Endocrinology \| www.frontiersin.org REFERENCES 10. Dralle H, Musholt TJ, Schabram J, Steinmüller T, Frilling A, Simon D, et al. German Association of Endocrine Surgeons Practice Guideline for the Surgical Management of Malignant Thyroid Tumors. Langenbeck's Arch Surg (2013) 398(3):347–75. doi: 10.1007/s00423-013-1057-6 1. Jin M, Kim ES, Kim BH, Kim HK, Kang YE, Jeon MJ, et al. Clinicopathological Characteristics and Disease-Free Survival in Patients With Hürthle Cell Carcinoma: A Multicenter Cohort Study in South Korea. Endocrinol Metab (2021) 36(5):1078–85. doi: 10.3803/EnM.2021.1151 11. Lopez-Penabad L, Chiu AC, Hoff AO, Schultz P, Gaztambide S, Ordoñez NG, et al. Prognostic Factors in Patients With Hürthle Cell Neoplasms of the Thyroid. Cancer (2003) 97(5):1186–94. doi: 10.1002/cncr.11176 2. Matsuura D, Yuan A, Wang L, Ranganath R, Adilbay D, Harries V, et al. Follicular and Hurthle Cell Carcinoma: Comparison of Clinicopathological Features and Clinical Outcomes. Thyroid (2022) 32(3):245–54. doi: 10.1089/ thy.2021.0424 12. Carcangiu ML. Hürthle Cell Carcinoma: Clinic-Pathological and Biological Aspects. Tumori (2003) 89(5):529–32. doi: 10.1177/ 030089160308900514 13. Kushchayeva Y, Duh QY, Kebebew E, Clark OH. Prognostic Indications for Hürthle Cell Cancer. World J Surg (2004) 28(12):1266–70. doi: 10.1007/ s00268-004-7602-2 3. LIoyd R, Osamura R, Kloppel G, Rosai J. Who Classiﬁcation of Tumours of Endocrine Organs. Lyon: International Agency for Research on Cancer (2017). 14. Carling T, Udelsman R. Follicular Neoplasms of the Thyroid: What to Recommend. Thyroid (2005) 15(6):583–7. doi: 10.1089/thy.2005.15.583 4. Phitayakorn R, McHenry CR. Follicular and Hürthle Cell Carcinoma of the Thyroid Gland. Surg Oncol Clin N Am (2006) 15(3):603–23, ix-x. doi: 10. 1016/j.soc.2006.05.011 15. Besic N, Hocevar M, Zgajnar J, Petric R, Pilko G. Aggressiveness of Therapy and Prognosis of Patients With Hürthle Cell Papillary Thyroid Carcinoma. Thyroid (2006) 16(1):67–72. doi: 10.1089/thy.2006.16.67 5. Khokar AM, Holoubek SA, Kuchta KM, Winchester DJ, Prinz RA, Moo- Young TA. Survival With Follicular and Hurthle Cell Microcarcinoma Compared to Papillary Thyroid Microcarcinoma: A Population Study of 84,532 Patients. World J Surg (2020) 44(2):469–78. doi: 10.1007/s00268-019- 05264-9 16. Montone KT, Baloch ZW, LiVolsi VA. The Thyroid Hürthle (Oncocytic) Cell and Its Associated Pathologic Conditions: A Surgical Pathology and Cytopathology Review. Arch Pathol Lab Med (2008) 132(8):1241–50. doi: 10.5858/2008-132-1241-tthoca 6. Nagar S, Aschebrook-Kilfoy B, Kaplan EL, Angelos P, Grogan RH. Hurthle Cell Carcinoma: An Update on Survival Over the Last 35 Years. Surgery (2013) 154(6):1263–71; discussion 71. doi: 10.1016/j.surg.2013.06.029 17. Guerrero MA, Suh I, Vriens MR, Shen WT, Gosnell J, Kebebew E, et al. DISCUSSION A recent retrospective analysis of the National Cancer Database (NCDB) suggested improved survival for HCC patients, who receive radioiodine treatment (27). Whether adjuvant external beam radiation therapy (EBRT) might be useful in these patients is not fully clear to date (28). There is a lack of evidence on EBRT for thyroid cancer recurrence. Salvage surgery for loco regional relapse can deliver information on the nature of recurrence. In this study, it was performed in 7 (13.7%) patients and revealed soft tissue metastases instead of lymph node metastases (Figure 1). This result is consistent with Bishop et al. (25), who analyzed 24 metastases and reported that there were no true lymphatic metastases in the majority of cases, but tumor emboli in veins that reimplant along the venous outﬂow tract. These metastatic nodules were not surrounded by any peripheral cuff of lymphoid Five (9.8%) patients with progressive metastatic disease were not referred to surgery in this study. Two of these patients agreed to tyrosine kinase treatment but treatment had to be changed or discontinued due to side effects. Both patients died two and four years after diagnosis respectively. A particular challenge for systemic treatment is the general lack of actionable somatic alterations in HCC (29). Alterations amenable to targeted therapy seem to be rare (30, 31). June 2022 \| Volume 13 \| Article 904986 5 Chiapponi et al. Chiapponi et al. Prophylactic Lymphadenectomy in HCC ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics board of the University of Cologne 50931 Cologne. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Since most of the patients in this study underwent a central lymphadenectomy, one might speculate on whether this may have contributed to the absence of lymph nodes in the loco-regional recurrences. However, the data of Coca Pelaz et al. (26) and those of Bishop et al. (25) also suggest that lymph nodal involvement is a rather rare event and most recurrences consist of soft tissue implants. Based on the present data, on the literature and on our clinical experience, it appears that prophylactic central lymphadenectomy does not avoid recurrence, which mostly consists of soft tissue metastasis, thus suggesting hematogenous rather than lymphogenous spread. Current evidence does not appear sufﬁcient for recommending routine central lymph node dissection for HCC, especially in the absence of vascular invasion. AUTHOR CONTRIBUTIONS Conceptualization, CC, HA, and MS; methodology and software CC; formal analysis, HA, MF, MS, MH, CB and AS; investigation, CC and MH; resources, MH, MS; data curation, CC; writing—original draft preparation CC, HA and MH; writing—review and editing, MS, MF, AS, and CB. All authors have read and agreed to the published version of the manuscript. DATA AVAILABILITY STATEMENT It needs to be mentioned that not all pathologic specimens were available for review. In some cases, only external pathology reports were used for diagnosis and treatment. Despite a new WHO classiﬁcation of Hürthle cell tumors though, 75% Hürthle cells in specimen was also required before 2017 for Hürthle cell diagnosis. It also needs to be mentioned the high persistence/ recurrence rate reported (23.5%), might be even higher, given an average time to relapse of 90.7 months, as reported in some studies (2, 32, 33). Finally, due to the rarity of HCC, the collective included is not large enough for formulating general recommendations. However, the data presented, and especially the rate of lymph node involvement are similar to those in the meta-analysis of Coca Pelaz et al. (26). The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. REFERENCES Age and Tumor Size Predicts Lymph Node Involvement in Hürthle Cell Carcinoma. J Cancer (2010) 1:23–6. doi: 10.7150/jca.1.23 7. Khaﬁf A, Khaﬁf RA, Attie JN. Hürthle Cell Carcinoma: A Malignancy of Low- Grade Potential. 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Pitoia F, Jerkovich F. Dynamic Risk Assessment in Patients With Differentiated Thyroid Cancer. Endocrine-Related Cancer (2019) 26(10): R553–R66. doi: 10.1530/erc-19-0213 June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org 6 Prophylactic Lymphadenectomy in HCC Chiapponi et al. Wnt/b-Catenin Pathway. Eur J Endocrinol (2020) 183(6):647–56. doi: 10. 1530/eje-20-0597 21. Chiapponi C, Hartmann MJM, Schmidt M, Faust M, Schultheis AM, Bruns CJ, et al. Radioiodine Refractory Follicular Thyroid Cancer and Surgery for Cervical Relapse. Cancers (Basel) (2021) 13(24):6230. doi: 10.3390/cancers13246230 31. Dong Y, Gong Y, Kuo F, Makarov V, Reznik E, Nanjangud GJ, et al. Targeting the Mtor Pathway in Hurthle Cell Carcinoma Results in Potent Antitumor Activity. Mol Cancer Ther (2022) 21(2):382–94. doi: 10.1158/1535-7163.Mct- 21-0224 22. Simões-Pereira J, Mourinho N, Ferreira TC, Limbert E, Cavaco BM, Leite V. Avidity and Outcomes of Radioiodine Therapy for Distant Metastasis of Distinct Types of Differentiated Thyroid Cancer. J Clin Endocrinol Metab (2021) 106(10):e3911–e22. doi: 10.1210/CLINEM/DGAB436 32. Kure S, Ohashi R. Thyroid Hürthle Cell Carcinoma: Clinical, Pathological, and Molecular Features. Cancers (Basel) (2020) 13(1):26. doi: 10.3390/ cancers13010026 23. Goffredo P, Roman SA, Sosa JA. Hurthle Cell Carcinoma: A Population-Level Analysis of 3311 Patients. Cancer (2013) 119(3):504–11. doi: 10.1002/ cncr.27770 33. Mills SC, Haq M, Smellie WJ, Harmer C. Hürthle Cell Carcinoma of the Thyroid: Retrospective Review of 62 Patients Treated at the Royal Marsden Hospital Between 1946 and 2003. REFERENCES Eur J Surg Oncol J Eur Soc Surg Oncol Br Assoc Surg Oncol (2009) 35(3):230–4. doi: 10.1016/j.ejso.2008.06.007 24. Chindris AM, Casler JD, Bernet VJ, Rivera M, Thomas C, Kachergus JM, et al. Clinical and Molecular Features of Hürthle Cell Carcinoma of the Thyroid. J Clin Endocrinol Metab (2015) 100(1):55–62. doi: 10.1210/jc.2014-1634 25. Bishop JA, Wu G, Tufano RP, Westra WH. Histological Patterns of Locoregional Recurrence in Hürthle Cell Carcinoma of the Thyroid Gland. Thyroid (2012) 22(7):690–4. doi: 10.1089/thy.2011.0407 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. 26. Coca-Pelaz A, Rodrigo JP, Shah JP, Sanabria A, Al Ghuzlan A, Silver CE, et al. Hürthle Cell Carcinoma of the Thyroid Gland: Systematic Review and Meta- Analysis. Adv Ther (2021) 38(10):5144–64. doi: 10.1007/s12325-021-01876-7 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. 27. Jillard CL, Youngwirth L, Scheri RP, Roman S, Sosa JA. Radioactive Iodine Treatment Is Associated With Improved Survival for Patients With Hürthle Cell Carcinoma. Thyroid (2016) 26(7):959–64. doi: 10.1089/thy.2016.0246 28. Ford D, Giridharan S, McConkey C, Hartley A, Brammer C, Watkinson JC, et al. External Beam Radiotherapy in the Management of Differentiated Thyroid Cancer. Clin Oncol (R Coll Radiol) (2003) 15(6):337–41. doi: 10. 1016/s0936-6555(03)00162-6 Copyright © 2022 Chiapponi, Hartmann, Schmidt, Faust, Bruns, Schultheis and Alakus. 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. 29. McFadden DG, Sadow PM. Genetics, Diagnosis, and Management of Hürthle Cell Thyroid Neoplasms. Front Endocrinol (Lausanne) (2021) 12:696386. doi: 10.3389/fendo.2021.696386 30. Santana NO, Lerario AM, Schmerling CK, Marui S, Alves VAF, Hoff AO, et al. June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org REFERENCES Molecular Proﬁle of Hürthle Cell Carcinomas: Recurrent Mutations in the June 2022 \| Volume 13 \| Article 904986 Frontiers in Endocrinology \| www.frontiersin.org

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